T-cell redirecting bispecific antibodies for treatment of disease

ABSTRACT

The present invention concerns compositions and methods of use of T-cell redirecting complexes, with at least one binding site for a T-cell antigen and at least one binding site for an antigen on a diseased cell or pathogen. Preferably, the complex is a DNL™ complex. More preferably, the complex comprises a bispecific antibody (bsAb). Most preferably, the bsAb is an anti-CD3×anti-CD19 bispecific antibody, although antibodies against other T-cell antigens and/or disease-associated antigens may be used. The complex is capable of targeting effector T cells to induce T-cell-mediated cytotoxicity of cells associated with a disease, such as cancer, autoimmune disease or infectious disease. The cytotoxic immune response is enhanced by co-administration of interferon-based agents that comprise interferon-α, interferon-β, interferon-λ1, interferon-λ2 or interferon-λ3.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) ofprovisional U.S. Patent Applications 61/682,965, filed Aug. 14, 2012;61/733,268, filed Dec. 4, 2012, and 61/807,998, filed Apr. 3, 2013, eachpriority application incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 9, 2013, isnamed IBC138US1_SL.txt and is 49,172 bytes in size.

FIELD

The present invention concerns compositions and methods of use of T-cellredirecting complexes. Preferably, the complexes comprise bispecificantibodies with one binding site for a T-cell antigen and anotherbinding site for an antigen expressed on a diseased cell or pathogen.However, in alternative embodiments a different type of binding moleculemay be utilized. In more preferred embodiments, the complexes are madeas DOCK-AND-LOCK™ complexes, in which the components are attachedtogether using the binding interaction between dimerization and dockingdomain (DDD) moieties from human protein kinase A (PKA) regulatorysubunits and anchor domain (AD) moieties from AKAPs (A-kinase anchorproteins). However, other methods of making bispecific antibodycomplexes are known and may be used. The subject complexes may compriseone or more antibodies or antigen-binding antibody fragments that bindto an antigen expressed on T cells, such as CD3, and one or moreantibodies or antibody fragments that bind to an antigen on a targetcell, such as CD19, CD20, CD22, CD33, CD66e, EpCAM, HER2/neu, EGFreceptor or another tumor-associated antigen (TAA), or an antigenexpressed on a different diseased cell or pathogenic organism. Thebispecific antibody redirects effector T cells to target diseased cells,tissues or pathogens and induces an immune response against the target.In more preferred embodiments, the bispecific antibody may be combinedwith one or more therapeutic agents that enhance the immune response.Most preferably, the therapeutic agent is an interferon, such asinterferon-α, interferon-β or interferon-λ. The subject compositions andmethods are of use for treating a wide variety of diseases and medicalconditions, such as cancer, autoimmune disease, immune systemdysfunction, graft-versus-host disease, organ transplant rejection orinfectious disease.

BACKGROUND

Use of bispecific antibodies (bsAbs) to redirect effector T cells forthe targeted killing of tumor cells has shown considerable promise bothpre-clinically and clinically (see, e.g., Topp et al., 2012, Blood120:5185-87; Bargou et al., 2008, Science 321:974-77). The bispecificantibodies contain a first binding site specific to CD3 for T-cellrecruitment and activation and a second binding site for a targeteddisease-associated antigen, such as CD19 (Bassan, 2012, Blood120:5094-95). The bispecific antibody brings CD3⁺ T cells into directcontact with targeted disease cells and induces cell-mediatedcytotoxicity (Bassan, 2012). Anti-CD3×anti-CD19 bispecific antibodieshave been reported to produce a complete and durable molecular remissionat very low concentrations in approximately 70% of adult patients withMRD⁺ ALL (Topp et al., 2012, Blood 120:5185-87). Bispecific antibodiesrecognizing gliomas and the CD3 epitope on T cells have beensuccessfully used in treating brain tumors in human patients (Nitta, etal. Lancet 1990; 355:368-371).

Numerous methods to produce bispecific antibodies are known (see, e.g.U.S. Pat. No. 7,405,320). Bispecific antibodies can be produced by thequadroma method, which involves the fusion of two different hybridomas,each producing a monoclonal antibody recognizing a different antigenicsite (Milstein and Cuello, Nature 1983; 305:537-540). The fusedhybridomas are capable of synthesizing two different heavy chains andtwo different light chains, which can associate randomly to give aheterogeneous population of 10 different antibody structures of whichonly one of them, amounting to ⅛ of the total antibody molecules, willbe bispecific, and therefore must be further purified from the otherforms. Fused hybridomas are often less stable cytogenetically than theparent hybridomas, making the generation of a production cell line moreproblematic.

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies, so that the resulting hybrid conjugate will bindto two different targets (Staerz, et al. Nature 1985; 314:628-631;Perez, et al. Nature 1985; 316:354-356). Bispecific antibodies generatedby this approach are essentially heteroconjugates of two IgG molecules,which diffuse slowly into tissues and are rapidly removed from thecirculation. Bispecific antibodies can also be produced by reduction ofeach of two parental monoclonal antibodies to the respective halfmolecules, which are then mixed and allowed to reoxidize to obtain thehybrid structure (Staerz and Bevan. Proc Natl Acad Sci USA 1986;83:1453-1457). An alternative approach involves chemically cross-linkingtwo or three separately purified Fab′ fragments using appropriatelinkers. All these chemical methods are undesirable for commercialdevelopment due to high manufacturing cost, laborious productionprocess, extensive purification steps, low yields (<20%), andheterogeneous products.

Discrete V_(H) and V_(L) domains of antibodies produced by recombinantDNA technology may pair with each other to form a dimer (recombinant Fvfragment) with binding capability (U.S. Pat. No. 4,642,334). However,such non-covalently associated molecules are not sufficiently stableunder physiological conditions to have any practical use. Cognate V_(H)and V_(L) domains can be joined with a peptide linker of appropriatecomposition and length (usually consisting of more than 12 amino acidresidues) to form a single-chain Fv (scFv) with binding activity.Methods of manufacturing scFv-based agents of multivalency andmultispecificity by varying the linker length were disclosed in U.S.Pat. No. 5,844,094, U.S. Pat. No. 5,837,242 and WO 98/44001. Commonproblems that have been frequently associated with generating scFv-basedagents of multivalency and multispecificity are low expression levels,heterogeneous products, instability in solution leading to aggregates,instability in serum, and impaired affinity.

Several bispecific antibodies targeting CD3 and CD19 are in clinicaldevelopment. An scFv-based bispecific antibody construct, known as BITE®(Bispecific T-cell Engager) employs a single polypeptide containing 2antigen-binding specificities, each contributed by a cognate VH and VL,linked in tandem via a flexible linker (see, e.g., Nagorsen et al.,2009, Leukemia & Lymphoma 50:886-91; Amann et al., 2009, J Immunother32:453-64; Baeuerle and Reinhardt, 2009, Cancer Res 69:4941-44). Anotherbispecific antibody called DART® (Dual-Affinity Re-Targeting) utilizes adisulfide-stabilized diabody design (see, e.g., Moore et al., 2011,Blood 117:4542-51; Yeti et al., 2010, Arthritis Rheum 62:1933-43). BothBITE® and DART® exhibit fast blood clearance due to their small size(˜55 kDa), which requires frequent administration to maintaintherapeutic levels of the bispecific antibodies.

A need exists for methods and compositions to generate improvedbispecific antibody complexes with longer T_(1/2), betterpharmacokinetic properties, increased in vivo stability and/or improvedin vivo efficacy. A further need exists for compositions and methods toimprove the efficacy of anti-CD3 based bispecific antibodies fortherapeutic use in cancer and other diseases, for example byco-administering adjunct therapeutic agents, such as interferons, thatenhance the efficacy of the bispecific antibody constructs.

SUMMARY

The present invention relates to compositions and methods of use ofnovel, T-cell redirecting complexes. Preferably, the complexes comprisebispecific antibodies (bsAbs), preferably comprising an anti-CD3 scFv orother antibody fragment attached to a stabilized F(ab)₂ or otherantibody fragment. An exemplary design disclosed in the Examples belowcombined an anti-CD3 scFv with an anti-CD19 F(ab)₂ to form a constructdesignated (19)-3s, which specifically targeted B cells. Other bsAbscombining anti-CD3 with antibody fragments against othertumor-associated antigens, discussed in more detail below, are of use intargeted T cell immunotherapy of various solid tumors. The advantages ofthis design include bivalent binding to tumor cells, a larger size (˜130kDa) to preclude rapid renal clearance, and potent T-cell mediatedcytotoxicity. The bsAbs mediate the formation of immunological synapsesbetween T cells and cognate target cells, induce T-cell activation andproliferation in the presence of target cells, redirect potent T-cellmediated killing of target cells in vitro and inhibit growth of humantumors in vivo.

A preferred embodiment concerns the subject bispecific antibodiesproduced as trivalent DNL™ complexes, with longer T_(1/2), betterpharmacokinetic properties and increased in vivo stability. Methods forproduction and use of DNL™ complexes, comprising dimers of DDD moietiesfrom human PKA regulatory subunits RIα, RIβ, RIIα or RIIβ, bound to ADmoieties from AKAPs, are well known (see, e.g., U.S. Pat. Nos.7,550,143; 7,521,056; 7,534,866; 7,527,787; 7,666,400; 7,906,118;7,901,680; 8,003,111 and 8,034,352, the Examples section of eachincorporated herein by reference.) By attaching different effectormoieties, such as antibodies or antibody fragments, to the DDD and ADmoieties, DNL™ complexes comprising virtually any combination ofeffectors may be constructed and used.

The antibodies of use can be of various isotypes, preferably human IgG1,IgG2, IgG3 or IgG4, more preferably comprising human IgG1 hinge andconstant region sequences. The antibodies or fragments thereof can bechimeric human-mouse, humanized (human framework and murinehypervariable (CDR) regions), or fully human, as well as variationsthereof, such as half-IgG4 antibodies (referred to as “unibodies”), asdescribed by van der Neut Kolfschoten et al. (Science 2007;317:1554-1557). More preferably, the antibodies or fragments thereof maybe designed or selected to comprise human constant region sequences thatbelong to specific allotypes, which may result in reduced immunogenicitywhen administered to a human subject. Preferred allotypes foradministration include a non-G1m1 allotype (nG1 m1), such as G1m3,G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype is selectedfrom the group consisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

In various embodiments, the T-cell redirecting bispecific antibodies maybe administered to a subject for treatment of a condition. The skilledartisan will realize that any condition that may be treated with aT-cell redirecting bispecific antibodies may be treated with the subjectcompositions and methods. Exemplary conditions include, but are notlimited to, cancer, hyperplasia, neurodegenerative disease, Alzheimer'sdisease, cardiovascular disease, metabolic disease, vasculitis, viralinfection, fungal infection, bacterial infection, diabetic retinopathy,macular degeneration, autoimmune disease, edema, pulmonary hypertension,sepsis, myocardial angiogenesis, plaque neovascularization, restenosis,neointima formation after vascular trauma, telangiectasia, hemophiliacjoints, angiofibroma, fibrosis associated with chronic inflammation,lung fibrosis, deep venous thrombosis or wound granulation.

In particular embodiments, the bsAbs may be of use to treat autoimmunedisease, such as acute idiopathic thrombocytopenic purpura, chronicidiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, type 1diabetes, type 2 diabetes, Henoch-Schonlein purpura,post-streptococcalnephritis, erythema nodosurn, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjögren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

In certain embodiments, the bsAbs may be of use for therapy of cancer.It is anticipated that any type of tumor and any type of tumor antigenmay be targeted. Exemplary types of cancers that may be targeted includeacute lymphoblastic leukemia, acute myelogenous leukemia, biliarycancer, breast cancer, cervical cancer, chronic lymphocytic leukemia,chronic myelogenous leukemia, colorectal cancer, endometrial cancer,esophageal, gastric, head and neck cancer, Hodgkin's lymphoma, lungcancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiplemyeloma, renal cancer, ovarian cancer, pancreatic cancer, glioma,melanoma, liver cancer, prostate cancer, and urinary bladder cancer.However, the skilled artisan will realize that tumor-associated antigensare known for virtually any type of cancer.

Tumor-associated antigens that may be targeted include, but are notlimited to, alpha-fetoprotein (AFP), α-actinin-4, A3, antigen specificfor A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CAl25, CAMEL,CAP-1, carbonic anhydrase IX, CASP-8/m, CCL19, CCL21, CD1, CD1a, CD2,CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21,CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L,CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70,CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147,CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α,colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-Met, DAM,EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growthfactor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100,GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and itssubunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M,HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen,KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitoryfactor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, mCRP, MCP-1,MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16,MUM-1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD-1receptor, placental growth factor, p53, PLAGL2, prostatic acidphosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5,RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin,TRAIL receptors, TNF-α, Tn antigen, Thomson-Friedenreich antigens, tumornecrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen,complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2,bcl-6, Kras, an oncogene marker and an oncogene product (see, e.g.,Sensi et al., Clin Cancer Res 2006, 12:5023-32; Parmiani et al., JImmunol 2007, 178:1975-79; Novellino et al. Cancer Immunol Immunother2005, 54:187-207).

Exemplary antibodies that may be used in combination with an anti-CD3antibody or fragment thereof include, but are not limited to, hA19(anti-CD19, U.S. Pat. No. 7,109,304), hR1 (anti-IGF-1R, U.S. patentapplication Ser. No. 12/722,645, filed Mar. 12, 2010), hPAM4(anti-mucin, U.S. Pat. No. 7,282,567), hA20 (anti-CD20, U.S. Pat. No.7,251,164), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496),the Examples section of each cited patent or application incorporatedherein by reference. Alternative antibodies that may be attached to theanti-CD3 for treatment of various disease states include, but are notlimited to, abciximab (anti-glycoprotein IIb/IIIa), alemtuzumab(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab(anti-CD33), ibritumomab (anti-CD20), panitumumab (anti-EGFR), rituximab(anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-ErbB2),lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor),ipilimumab (anti-CTLA-4), abagovomab (anti-CA-125), adecatumumab(anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab (anti-CD125),obinutuzumab (GA101, anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026(anti-PSMA, U.S. patent application Ser. No. 11/983,372, deposited asATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575),tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab(anti-CD25), efalizumab (anti-CD11a), GA101 (anti-CD20; Glycart Roche),atalizumab (anti-α4 integrin), omalizumab (anti-IgE); anti-TNF-αantibodies such as CDP571 (Ofei et al., 2011, Diabetes 45:881-85),MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (Thermo Scientific,Rockford, Ill.), infliximab (Centocor, Malvern, Pa.), certolizumab pegol(UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels, Belgium),adalimumab (Abbott, Abbott Park, Ill.), BENLYSTA® (Human GenomeSciences); antibodies for therapy of Alzheimer's disease such as Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,solanezumab and infliximab; anti-fibrin antibodies like 59D8, T2G1s,MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202(Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson & Johnson);anti-HIV antibodies such as P4/D10 (U.S. Pat. No. 8,333,971), Ab 75, Ab76, Ab 77 (Paulik et al., 1999, Biochem Pharmacol 58:1781-90), as wellas the anti-HIV antibodies described and sold by Polymun (Vienna,Austria), also described in U.S. Pat. No. 5,831,034, U.S. Pat. No.5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos etal., Antimicrob. Agents Chemother. 2006; 50(5):1773-9.

In other embodiments, the subject bsAbs may be of use to treat subjectsinfected with pathogenic organisms, such as bacteria, viruses or fungi.Exemplary fungi that may be treated include Microsporum, Trichophyton,Epidermophyton, Sporothrix schenckii, Cryptococcus neoformans,Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidisor Candida albican. Exemplary viruses include human immunodeficiencyvirus (HIV), herpes virus, cytomegalovirus, rabies virus, influenzavirus, human papilloma virus, hepatitis B virus, hepatitis C virus,Sendai virus, feline leukemia virus, Reo virus, polio virus, human serumparvo-like virus, simian virus 40, respiratory syncytial virus, mousemammary tumor virus, Varicella-Zoster virus, dengue virus, rubellavirus, measles virus, adenovirus, human T-cell leukemia viruses,Epstein-Barr virus, murine leukemia virus, mumps virus, vesicularstomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus orblue tongue virus. Exemplary bacteria include Bacillus anthracis,Streptococcus agalactiae, Legionella pneumophilia, Streptococcuspyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseriameningitidis, Pneumococcus spp., Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis or aMycoplasma. Known antibodies against pathogens include, but are notlimited to, P4D10 (anti-HIV), CR6261 (anti-influenza), exbivirumab(anti-hepatitis B), felvizumab (anti-respiratory syncytial virus),foravirumab (anti-rabies virus), motavizumab (anti-respiratory syncytialvirus), palivizumab (anti-respiratory syncytial virus), panobacumab(anti-Pseudomonas), rafivirumab (anti-rabies virus), regavirumab(anti-cytomegalovirus), sevirumab (anti-cytomegalovirus), tivirumab(anti-hepatitis B), and urtoxazumab (anti-E. coli).

The subject bsAbs may be administered in combination with one or moreimmunomodulators to enhance the immune response. Immunomodulators mayinclude, but are not limited to, a cytokine, a chemokine, a stem cellgrowth factor, a lymphotoxin, an hematopoietic factor, a colonystimulating factor (CSF), erythropoietin, thrombopoietin, tumor necrosisfactor-α (TNF), TNF-β, granulocyte-colony stimulating factor (G-CSF),granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, interferon-λ, stem cell growth factordesignated “S1 factor”, human growth hormone, N-methionyl human growthhormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin,proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepaticgrowth factor, prostaglandin, fibroblast growth factor, prolactin,placental lactogen, OB protein, mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, NGF-β, platelet-growth factor, TGF-α, TGF-β,insulin-like growth factor-I, insulin-like growth factor-II,macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin,endostatin, or lymphotoxin. In certain embodiments, the bispecificantibody or antibody fragment may be attached to an immunomodulator,such as a cytokine. Cytokine complexes are disclosed, for example, inU.S. Pat. Nos. 7,906,118 and 8,034,352, the Examples section of eachincorporated herein by reference. Preferred immunomodulators that may beused in combination with T-cell redirecting bsAbs include interferon-α,interferon-β and interferon-λ.

Although the antibody or other binding molecule specific for effector Tcells preferably binds to the CD3 antigen, other antigens expressed oneffector T cells are known and may be targeted by the T-cell redirectingcomplex. Exemplary T-cell antigens include, but are not limited to, CD2,CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69and CD90.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1. Schematic diagram of formation of DOCK-AND-LOCK™ complexcomprising anti-CD19 F(ab)₂×anti-CD3 scFv.

FIG. 2. Immune synapse formation between Daudi Burkitt lymphoma and Tcells, mediated by (19)-3s. Freshly isolated T cells were combined withDaudi cells at an E:T ratio of 2.5:1. Cells were treated with 0, 1 or 5μg/mL of (19)-3s for 30 min at room temperature prior to analysis byflow cytometry. Anti-CD20-FITC and anti-CD7-APC were used to identifyDaudi and T cells, respectively. Co-binding was indicated as the % ofCD20⁺/CD7⁺ events. After treatment with (19)-3s, 45.5% of flow eventswere CD20/CD7 dual-positive, indicating synapsed Daudi and T cells (A),compared to 2% measured for the mixed cells without antibody (B).Addition of (19)-3s resulted in association of >90% of the Daudi with Tcells (C).

FIG. 3. Jurkat (T cells) and Daudi (B cells) were combined at a 1:1ratio, treated with 0.1 μg/mL (19)-3s for 30 minutes and stained withanti-CD20-FITC (A) and anti-CD3-PE (B), prior to analysis byfluorescence microscopy. The merged image (C) reveals synapse formationbetween green-stained Daudi and red-stained Jurkat cells. Synapseformation was not evident in the absence of (19)-3s (D).

FIG. 4. Dose response analysis of (19)-3s mediated cell-to-cellassociation of Daudi and Jurkat cells.

FIG. 5. Comparison of cell-to-cell association mediated by (A) BITE™,DART™ and (B) (19)-3s. The data for BITE™ and DART™ was taken from Mooreet al. (2011, Blood 117:4542-4551.

FIG. 6. Synapse formation between T cells and Capan-1 pancreatic cancercells mediated by (A) (19)-3s control bsAb compared to (B) (M1)-3sMUC5AC and (C) (E1)-3s TROP-2 targeting bsAbs. CFSE-labeled Capan-1cells were coincubated with PKH26-labeled Jurkat in the presence of thebsAbs.

FIG. 7. T-cell activation by (19)-3s. Upregulation of CD69 expression isan early event in T-cell activation. Daudi cells combined with PBMCs(A), or purified T cells (B), as well as purified T cells alone (C) weretreated overnight with the indicated antibodies, and stained withanti-CD3-PE and anti-CD69-APC, prior to analysis by flow cytometry. CD69expression was evaluated following gating of T cells by forward vs. sidescattering and anti-CD3 staining. (A) Combination of Daudi cells with anequal number of PBMCs resulted in 1.6% CD69+ T cells. Addition of 3ng/mL (19)-3s induced 27% CD69+ T cells. Neither a control construct[(M1)-3s], which comprises the Okt3-scFv-AD2 module fused with anon-targeting F(ab)₂, nor the hA19-Fab-DDD2 module, induced T-cellactivation. (B) Treatment of Daudi and purified T cells with (M1)-3s orhA19-Fab-DDD2 did not increase the number of CD69+ T cells (<4%),compared to the untreated cell mixture. Alternatively, (19)-3s inducedrobust T-cell activation, producing 80% CD69+ cells. (C) Without theaddition of Daudi (target) cells, (19)-3s did not induce CD69 expressionand T-cell activation. These results demonstrate that (19)-3s-mediatedsynapse formation between T cells and target cells is both required andsufficient for T-cell activation.

FIG. 8. Induction of T-cell proliferation by (19)-3s. (A) PBMCs wereincubated with 3 nM or 30 pM of (19)-3s, compared to IL-2/PHA positivecontrol and (14)-3s (non-target-binding control). (B) T cellproliferation was not observed in PBMCs depleted of B cells, indicatingthat target cells (B cells) are required for T-cell activation andproliferation.

FIG. 9. In vitro cytotoxicity of (19)-3s T-cell redirecting bsAbs.Dose-response curves for cytotoxicity to Nalm-6, Raji, Ramos and Namalwacancer cells were determined for the (A) (19)-3s and (B) (14)-3s(non-targeting) DNL™ bsAb complexes. (C) Consistent results wereobserved using PBMCs, or T cells, obtained from two different donors andNalm-6 cancer cells.

FIG. 10. In vitro cytotoxicity of (20)-3s, (22)-3s and (C2)-3s T-cellredirecting bsAbs. Dose-response curves were determined for cytotoxicityto (A) Namalwa, (B) Jeko, and (C) Daudi cells induced by (20)-3s,(22)-3s and (C2)-3s T-cell redirecting bsAbs.

FIG. 11. In vitro cytotoxicity of T-cell redirecting bsAbs in solidtumor cell lines. (A) Dose-response curves were determined forcytotoxicity to the LS174T colon adenocarcinoma cell line for the(14)-3s bsAb, compared to non-targeting (19)-3s bsAb. (B) Dose-responsecurves were determined for cytotoxicity to the Capan-1 pancreaticadenocarcinoma cell line for the (E1)-3s bsAb, compared to non-targeting(19)-3s bsAb. (C) Dose-response curves were determined for cytotoxicityto the NCI-N87 gastric carcinoma cell line for the (E1)-3s and (15)-3sbsAbs, compared to non-targeting (19)-3s bsAb.

FIG. 12. Summary of in vitro cytotoxicity data for T-cell redirectingbsAbs in cancer cell lines.

FIG. 13. In vivo retargeting of Raji lymphoma xenografts using (19)-3sbsAb. NOD/SCID mice bearing Raji Burkitt lymphoma (1×10⁶ cells)xenografts, reconstituted with human PBMCs (5×10⁶ cells) were treatedwith (19)-3s for only 1 week, administered as indicated by the arrows:(A) untreated, (B) treated with a single dose of 130 μg, (C) treated 3×with 43 μg per dose, (D) treated 5× with 26 μg per dose.

FIG. 14. Effect of repeated dosing on in vivo retargeting of Rajilymphoma xenografts using (19)-3s bsAb. NOD/SCID mouse xenografts wereprepared as indicated in the legend to FIG. 13. The (19)-3s wasadministered as indicated by the arrows: (A) untreated, (B) treated 2×with 130 μg per dose of (19)-3s administered i.v., (C) treated 2× with130 μs per dose of (19)-3s administered s.c., (D) treated 4× with 65 μgper dose of (19)-3s administered i.v., (E) treated 6× with 43 μg perdose of (19)-3s administered i.v., (F) treated 6× with 43 μg per dose ofcontrol (M1)-3s administered i.v.

FIG. 15. In vivo efficacy of T-cell retargeting bsAbs in solid tumorxenografts. NOD/SCID mouse xenografts were prepared with LS174T colonadenocarcinoma (A, B) or Capan-1 pancreatic carcinoma (C, D). (A) Micewere administered T cells only without bsAb. (B) Mice were treated with(E1)-3s bsAb as indicated. (C) Mice were administered PBMCs only withoutbsAb. (D) Mice were treated with (14)-3s bsAb as indicated.

FIG. 16. In vivo inhibition of tumor growth by (E1)-3s DNL™ complex inthe presence or absence of interferon-α. Capan-1 pancreatic carcinomaxenografts in NOD/SCID mice were treated with anti-TROP-2×anti-CD3 bsAbwith or without added interferon-α. (A) The interferon-α was added inthe form of a TROP-2 targeting DNL™ complex. (B) The interferon-α wasadded as the commercially available PEGASYS® (peginterferon alfa-2a).

FIG. 17. Survival curves for NOD/SCID mice treated with (E1)-3s with orwithout interferon-α. Controls were untreated or treated withinterferon-α alone.

DETAILED DESCRIPTION

Definitions

Unless otherwise specified, “a” or “an” means “one or more”.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, peptides, drugs, toxins, enzymes,nucleases, hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,dyes, and radioisotopes.

An “antibody” as used herein refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment. An“antibody” includes monoclonal, polyclonal, bispecific, multispecific,murine, chimeric, humanized and human antibodies.

A “naked antibody” is an antibody or antigen binding fragment thereofthat is not attached to a therapeutic or diagnostic agent. The Fcportion of an intact naked antibody can provide effector functions, suchas complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev50:59-87, 1998). Other mechanisms by which naked antibodies induce celldeath may include apoptosis. (Vaswani and Hamilton, Ann Allergy AsthmaImmunol 81: 105-119, 1998.)

An “antibody fragment” is a portion of an intact antibody such asF(ab′)₂, F(ab)₂, Fab′, Fab, Fv, scFv, dAb and the like. Regardless ofstructure, an antibody fragment binds with the same antigen that isrecognized by the full-length antibody. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains or recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). “Single-chain antibodies”, often abbreviated as“scFv” consist of a polypeptide chain that comprises both a V_(H) and aV_(L) domain which interact to form an antigen-binding site. The V_(H)and V_(L) domains are usually linked by a peptide of 1 to 25 amino acidresidues. Antibody fragments also include diabodies, triabodies andsingle domain antibodies (dAb).

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains, including human framework region(FR) sequences. The constant domains of the antibody molecule arederived from those of a human antibody. To maintain binding activity, alimited number of FR amino acid residues from the parent (e.g., murine)antibody may be substituted for the corresponding human FR residues.

A “human antibody” is an antibody obtained from transgenic mice thathave been genetically engineered to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A human antibodyalso can be constructed by genetic or chromosomal transfection methods,as well as phage display technology, all of which are known in the art.(See, e.g., McCafferty et al., 1990, Nature 348:552-553 for theproduction of human antibodies and fragments thereof in vitro, fromimmunoglobulin variable domain gene repertoires from unimmunizeddonors). In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see, e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.(See, U.S. Pat. Nos. 5,567,610 and 5,229,275).

As used herein, the term “antibody fusion protein” is a recombinantlyproduced antigen-binding molecule in which an antibody or antibodyfragment is linked to another protein or peptide, such as the same ordifferent antibody or antibody fragment or a DDD or AD peptide. Thefusion protein may comprise a single antibody component, a multivalentor multispecific combination of different antibody components ormultiple copies of the same antibody component. The fusion protein mayadditionally comprise an antibody or an antibody fragment and atherapeutic agent. Examples of therapeutic agents suitable for suchfusion proteins include immunomodulators and toxins. One preferred toxincomprises a ribonuclease (RNase), preferably a recombinant RNase. Apreferred immunomodulator might be an interferon, such as interferon-α,interferon-β or interferon-λ.

A “multispecific antibody” is an antibody that can bind simultaneouslyto at least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. A “multivalent antibody” is anantibody that can bind simultaneously to at least two targets that areof the same or different structure. Valency indicates how many bindingarms or sites the antibody has to a single antigen or epitope; i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyis able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Multispecific, multivalent antibodiesare constructs that have more than one binding site of differentspecificity.

A “bispecific antibody” is an antibody that can bind simultaneously totwo targets which are of different structure. Bispecific antibodies(bsAb) and bispecific antibody fragments (bsFab) may have at least onearm that specifically binds to, for example, a T cell, and at least oneother arm that specifically binds to an antigen produced by orassociated with a diseased cell, tissue, organ or pathogen, for examplea tumor-associated antigen. A variety of bispecific antibodies can beproduced using molecular engineering.

An antibody preparation, or a composition described herein, is said tobe administered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient subject. In particular embodiments, anantibody preparation is physiologically significant if its presenceinvokes an antitumor response or mitigates the signs and symptoms of anautoimmune or infectious disease state. A physiologically significanteffect could also be the evocation of a humoral and/or cellular immuneresponse in the recipient subject leading to growth inhibition or deathof target cells.

Interferon Therapy

In various embodiments, the subject T-cell redirecting bsAbs may be usedin combination with one or more interferons, such as interferon-α,interferon-β or interferon-λ. Human interferons are well known in theart and the amino acid sequences of human interferons may be readilyobtained from public databases (e.g., GenBank Accession Nos. AAA52716.1;AAA52724; AAC41702.1; EAW56871.1; EAW56870.1; EAW56869.1). Humaninterferons may also be commercially obtained from a variety of vendors(e.g., Cell Signaling Technology, Inc., Danvers, Mass.; Genentech, SouthSan Francisco, Calif.; EMD Millipore, Billerica, Mass.).

Interferon-α (IFNα) has been reported to have anti-tumor activity inanimal models of cancer (Ferrantini et al., 1994, J Immunol 153:4604-15)and human cancer patients (Gutterman et al., 1980, Ann Intern Med93:399-406). IFNα can exert a variety of direct anti-tumor effects,including down-regulation of oncogenes, up-regulation of tumorsuppressors, enhancement of immune recognition via increased expressionof tumor surface MHC class I proteins, potentiation of apoptosis, andsensitization to chemotherapeutic agents (Gutterman et al., 1994, PNASUSA 91:1198-205; Matarrese et al., 2002, Am J Pathol 160:1507-20;Mecchia et al., 2000, Gene Ther 7:167-79; Sabaawy et al., 1999, Int JOncol 14:1143-51; Takaoka et al, 2003, Nature 424:516-23). For sometumors, IFNα can have a direct and potent anti-proliferative effectthrough activation of STAT1 (Grimley et al., 1998 Blood 91:3017-27).Interferon-α2b has been conjugated to anti-tumor antibodies, such as thehL243 anti-HLA-DR antibody and depletes lymphoma and myeloma cells invitro and in vivo (Rossi et al., 2011, Blood 118:1877-84).

Indirectly, IFNα can inhibit angiogenesis (Sidky and Borden, 1987,Cancer Res 47:5155-61) and stimulate host immune cells, which may bevital to the overall antitumor response but has been largelyunder-appreciated (Belardelli et al., 1996, Immunol Today 17:369-72).IFNα has a pleiotropic influence on immune responses through effects onmyeloid cells (Raefsky et al, 1985, J Immunol 135:2507-12; Luft et al,1998, J Immunol 161:1947-53), T-cells (Carrero et al, 2006, J Exp Med203:933-40; Pilling et al., 1999, Eur J Immunol 29:1041-50), and B-cells(Le et al, 2001, Immunity 14:461-70). As an important modulator of theinnate immune system, IFNα induces the rapid differentiation andactivation of dendritic cells (Belardelli et al, 2004, Cancer Res64:6827-30; Paquette et al., 1998, J Leukoc Biol 64:358-67; Santini etal., 2000, J Exp Med 191:1777-88) and enhances the cytotoxicity,migration, cytokine production and antibody-dependent cellularcytotoxicity (ADCC) of NK cells (Biron et al., 1999, Ann Rev Immunol17:189-220; Brunda et al. 1984, Cancer Res 44:597-601).

Interferon-β has been reported to be efficacious for therapy of avariety of solid tumors. Patients treated with 6 million units of IFN-βtwice a week for 36 months showed a decreased recurrence ofhepatocellular carcinoma after complete resection or ablation of theprimary tumor in patients with HCV-related liver cancer (Ikeda et al.,2000, Hepatology 32:228-32). Gene therapy with interferon-β inducedapoptosis of glioma, melanoma and renal cell carcinoma (Yoshida et al.,2004, Cancer Sci 95:858-65). Endogenous IFN-β has been observed toinhibit tumor growth by inhibiting angiogenesis in vivo (Jablonska etal., 2010, J Clin Invest. 120:1151-64.)

The therapeutic effectiveness of IFNs has been validated to date by theapproval of IFN-α2 for treating hairy cell leukemia, chronic myelogenousleukemia, malignant melanoma, follicular lymphoma, condylomataacuminata, AIDs-related Kaposi sarcoma, and chronic hepatitis B and C;IFN-β for treating multiple sclerosis; and IFN-γ for treating chronicgranulomatous disease and malignant osteopetrosis. Despite a vastliterature on this group of autocrine and paracrine cytokines, theirfunctions in health and disease are still being elucidated, includingmore effective and novel forms being introduced clinically (Pestka,2007, J. Biol. Chem. 282:20047-51; Vilcek, 2006, Immunity 25:343-48).

Interferons are critical role players in the antitumor and antimicrobialhost defense, and have been extensively explored as therapeutic agentsfor cancer and infectious disease (Billiau et al., 2006, Cytokine GrowthFactor Rev 17:381-409; Pestka et al., 2004, Immunol Rev 202:8-32).Despite considerable efforts with type I and II interferons (IFN-α/β andγ), their use in clinic settings have been limited because of the shortcirculation half-life, systemic toxicity, and suboptimal responses inpatients (Pestka et al., 2004, Immunol Rev 202:8-32; Miller et al.,2009, Ann NY Acad Sci 1182:69-79). The discovery of the IFN-λ family inearly 2003 brought an exciting new opportunity to develop alternativeIFN agents for these unmet clinical indications (Kotenko et al., 2003,Nat Immunol 4:69-77; Sheppard et al., 2003, Nat Immunol 4:63-8).

IFN-λs, designated as type III interferons, are a newly described groupof cytokines that consist of IFN-λ1, 2, 3 (also referred to asinterleukin-29, 28A, and 28B, respectively), that are geneticallyencoded by three different genes located on chromosome 19 (Kotenko etal., 2003, Nat Immunol 4:69-77; Sheppard et al., 2003, Nat Immunol4:63-8). At the protein level, IFN-λ2 and -λ3 are is highly homologous,with 96% amino acid identity, while IFN-λ1 shares approximately 81%homology with IFN-λ2 and -λ3 (Sheppard et al., 2003, Nat Immunol4:63-8). IFN-λs activate signal transduction via the JAK/STAT pathwaysimilar to that induced by type I IFN, including the activation of JAK1and TYK2 kinases, the phosphorylation of STAT proteins, and theactivation of the transcription complex of IFN-stimulated gene factor 3(ISGF3) (Witte et al., 2010, Cytokine Growth Factor Rev 21:237-51; Zhouet al., 2007, J Virol 81:7749-58).

A major difference between type III and type I IFN systems is thedistribution of their respective receptor complexes. IFN-α/β signalsthrough two extensively expressed type I interferon receptors, and theresulting systemic toxicity associated with IFN-α/β administration haslimited their use as therapeutic agents (Pestka et al., 2007, J BiolChem 282:20047-51). In contrast, IFN-λs signal through a heterodimericreceptor complex consisting of unique IFN-λ receptor 1 (IFN-λR1) andIL-10 receptor 2 (IL-10R2). As previously reported (Witte et al., 2009,Genes Immun 10:702-14), IFN-λR1 has a very restricted expression patternwith the highest levels in epithelial cells, melanocytes, andhepatocytes, and the lowest level in primary central nervous system(CNS) cells. Blood immune system cells express high levels of a shortIFN-λ receptor splice variant (sIFN-λR1) that inhibits IFN-λ action. Thelimited responsiveness of neuronal cells and immune cells implies thatthe severe toxicity frequently associated with IFN-α therapy may beabsent or significantly reduced with IFN-λs (Witte et al., 2009, GenesImmun 10:70244; Witte et al., 2010, Cytokine Growth Factor Rev21:237-51). A recent publication reported that while IFN-α and IFN-λinduce expression of a common set of ISGs (interferon-stimulated genes)in hepatocytes, unlike IFN-α, administration of IFN-λ did not induceSTAT activation or ISG expression in purified lymphocytes or monocytes(Dickensheets et al., 2013, J Leukoc Biol. 93, published online Dec. 20,2012). It was suggested that IFN-λ may be superior to IFN-α fortreatment of chronic HCV infection, as it is less likely to induceleukopenias that are often associated with IFN-α therapy (Dickensheetset al., 2013).

IFN-λs display structural features similar to IL-10-related cytokines,but functionally possess type I IFN-like anti-viral andanti-proliferative activity (Witte et al., 2009, Genes Immun 10:702-14;Ank et al., 2006, J Virol 80:4501-9; Robek et al., 2005, J Virol79:3851-4). IFN-λ1 and -λ2 have been demonstrated to reduce viralreplication or the cytopathic effect of various viruses, including DNAviruses (hepatitis B virus (Robek et al., 2005, J Virol 79:3851-4, Doyleet al., 2006, Hepatology 44:896-906) and herpes simplex virus 2 (Ank etal., 2008, J Immunol 180:2474-85)), ss (+) RNA viruses (EMCV; Sheppardet al., 2003, Nat Immunol 4:63-8) and hepatitis C virus (Robek et al.,2005, J Virol 79:3851-4, Doyle et al., 2006, Hepatology 44:896-906;Marcello et al., 2006, Gastroenterol 131:1887-98; Pagliaccetti et al.,2008, J Biol Chem 283:30079-89), ss (−) RNA viruses (vesicularstomatitis virus; Pagliaccetti et al., 2008, J Biol Chem 283:30079-89)and influenza-A virus (Jewell et al., 2010, J Virol 84:11515-22) anddouble-stranded RNA viruses, such as rotavirus (Pott et al., 2011, PNASUSA 108:7944049). IFN-λ3 has been identified from genetic studies as akey cytokine in HCV infection (Ge et al., 2009, Nature 461:399-401), andhas also shown potent activity against EMCV (Dellgren et al., 2009,Genes Immun 10:125-31). A deficiency of rhinovirus-induced IFN-λproduction was reported to be highly correlated with the severity ofrhinovirus-induced asthma exacerbation (Controli et al., 2006, NatureMed 12:1023-26) and IFN-λ therapy has been suggested as a new approachfor treatment of allergic asthma (Edwards and Johnston, 2011, EMBO MolMed 3:306-8; Koltsida et al., 2011, EMBO Mol Med 3:348-61).

The anti-proliferative activity of IFN-λs has been established inseveral human cancer cell lines, including neuroendocrine carcinoma BON1(Zitzmann et al., 2006, Biochem Biophys Res Commun 344:1334-41),glioblastoma LN319 (Meager et al., 2005, Cytokine 31:109-18),immortalized keratinocyte HaCaT (Maher et al., 2008, Cancer Biol Ther7:1109-15), melanoma F01 (Guenterberg et al., 2010, Mol Cancer Ther9:510-20), and esophageal carcinoma TE-11 (Li et al., 2010, Eur J Cancer46:180-90). In animal models, IFN-λs induce both tumor apoptosis anddestruction through innate and adaptive immune responses, suggestingthat local delivery of IFN-λ might be a useful adjunctive strategy inthe treatment of human malignancies (Numasaki et al., 2007, J Immunol178:5086-98).

In clinical settings, PEGylated IFN-λ1 (PEG-IFN-λ1) has beenprovisionally used for patients with chronic hepatitis C virusinfection. In a phase Ib study (n=56), antiviral activity was observedat all dose levels (0.5-3.0 μg/kg), and viral load reduced 2.3 to 4.0logs when PEG-IFN-λ1 was administrated to genotype 1 HCV patients whorelapsed after IFN-α therapy (Muir et al., 2010, Hepatology 52:822-32).A phase IIb study (n=526) showed that patients with HCV genotypes 1 and4 had significantly higher response rates to treatment with PEG-IFN-λ1compared to PEG-IFN-α. At the same time, rates of adverse eventscommonly associated with type I interferon treatment were lower withPEG-IFN-λ1 than with PEG-IFN-α. Neutropenia and thrombocytopenia wereinfrequently observed and the rates of flu-like symptoms, anemia, andmusculoskeletal symptoms decreased to about ⅓ of that seen withPEG-IFN-α treatment. However, rates of serious adverse events,depression and other common adverse events (≧10%) were similar betweenPEG-IFN-λ1 and PEG-IFN-α. Higher rates of hepatotoxicity were seen inthe highest-dose PEG-IFN-λ1 compared with PEG-IFN-α (“InvestigationalCompound PEG-Interferon Lambda Achieved Higher Response Rates with FewerFlu-like and Musculoskeletal Symptoms and Cytopenias Than PEG-InterferonAlfa in Phase IIb Study of 526 Treatment-Naive Hepatitis C Patients,”Apr. 2, 2011, Press Release from Bristol-Myers Squibb).

In various embodiments, the subject T-cell redirecting bispecificantibodies may be used in combination with one or more interferons, suchas interferon-α, interferon-β, interferon-λ1, interferon-λ2, orinterferon-λ3. When used with the subject bsAbs, the interferon may beadministered prior to, concurrently with, or after the bsAb. Whenadministered concurrently, the interferon may be either conjugated to orseparate from the bsAb.

T-Cell Redirecting Bispecific Antibody Complexes

Various embodiments concern bsAbs comprising an anti-CD3 antibody orfragment thereof attached to an antibody or fragment thereof against adisease-associated antigen, such as CD19. Bispecific anti-CD3×anti-CD19antibodies are known in the art and are presently in clinicaldevelopment, such as BITE® (Bispecific T-cell Engager) (e.g., Nagorsenet al., 2009, Leukemia & Lymphoma 50:886-91; Amann et al., 2009, JImmunother 32:453-64; Baeuerle and Reinhardt, 2009, Cancer Res69:4941-44) and DART® (see, e.g., Moore et al., 2011, Blood 117:4542-51;Veri et al., 2010, Arthritis Rheum 62:1933-43).

Blinatumomab is a BITE® antibody comprising V_(H) and V_(L) domains ofanti-CD3 and anti-CD19 antibody fragments, connected with a 5-amino acidlinker and expressed as a single polypeptide chain that anneals toitself to form the antigen-binding sites. It is thought thatblinatumomab acts by bringing the T-cell-specific CD3 and B-cellspecific CD19 antigens into close proximity, to initiate a T-cellcytotoxic response against the juxtaposed B cells, which does notrequire T-cell specificity to the cancer cells (e.g., Portell et al.,2013, Clin Pharmacol 5(Suppl 1): 5-11). Due to its short half-life,blinatumomab requires continuous intravenous infusion to be effective,(Portell et al., 2013). A phase II trial of B-cell ALL patients withpersistent or relapsed minimal residual disease reported anapproximately 80% rate of complete remission (Portell et al., 2013).

Doses of blinatumomab as low as 0.005 mg/m²/day were reported to beeffective to eliminate cancer cells in non-Hodgkin's lymphoma patients(Bargou et al., 2008, Science 321:974-77). Partial and completeremissions were observed starting at a dose level of 0.015 mg and allsix patients tested at a dose of 0.06 mg experienced a tumor regression(Bargou et al., 2008). In vitro, blinatumomab induced 50% cell lysis ofMEC-1 cells at a concentration of 10 pg/mL (Topp et al., 2012, Blood120:5185-87; Bassan et al., 2012, Blood 120:5094-95).

The anti-CD19 portion of blinatumomab was derived from the HD37hybridoma (see, e.g., U.S. Pat. No. 7,575,923, the Examples section ofwhich is incorporated herein by reference), which is publicly available(e.g., Santa Cruz Biotechnology Cat. No. sc-18894). The anti-CD3 portionof blinatumomab was derived from the TR66 hybridoma (U.S. Pat. No.7,575,923; Traunecker et al., 1991, EMBO J. 10:3655-59), also publiclyavailable (e.g., Enzo Life Sciences, catalog No. ALX-804-822-C100).

A variety of antibodies against CD3 that may be used in the claimedmethods and compositions are publicly known and/or commerciallyavailable, such as from LSBio (catalog Nos. LS-B6698, LS-B8669;LS-B8765, LS-C96311, LS-058677, etc.); ABCAM® (catalog Nos. ab5690,ab16669, ab699, ab828, ab8671, etc.); Santa Cruz Biotechnology (catalogNos. sc-20047, sc-20080, sc-19590, sc-59008, sc-101442, etc.); and manyother suppliers.

In a preferred embodiment, the amino acid sequence of the anti-CD3moiety, used as part of a DNL™ complex, is as disclosed below in SEQ IDNO:96 to SEQ ID NO:101. However, the person of ordinary skill willrealize that any known anti-CD3 antibody may be utilized in the claimedmethods and compositions. Preferably, the antibody moieties of use arehumanized or human.

A variety of antibodies against CD19 that may be used in the claimedmethods and compositions are publicly known and/or commerciallyavailable, such as from Santa Cruz Biotechnology (catalog Nos.sc-390244, sc-373897, sc-18894, sc-18896, etc.); ABCAM® (catalog Nos.ab25232, ab134114, ab140981, ab1255, etc.); ABBIOTEC™ (catalog Nos.252262, 252248, 250585, 251063, etc.) and many other vendors.

In a preferred embodiment, the anti-CD19 antibody moiety is a humanizedA19 antibody, comprising the light chain CDR sequences CDR1KASQSVDYDGDSYLN (SEQ ID NO:90); CDR2 DASNLVS (SEQ ID NO:91); and CDR3QQSTEDPWT (SEQ ID NO:92) and the heavy chain CDR sequences CDR1 SYWMN(SEQ ID NO:93); CDR2 QIWPGDGDTNYNGKFKG (SEQ ID NO:94) and CDR3RETTTVGRYYYAMDY (SEQ ID NO:95).

Other anti-CD3×anti-CD19 bispecific antibodies are known, such as DART®,which also incorporates the anti-CD19 Fv sequences of HD37 and theanti-CD3 Fv sequences of TR66 (Moore et al., 2011, Blood 117:4542-51;Veri et al., 2010, Arthritis Rheum 62:1933-43). Moore et al. (2011)reported that DART® bispecific antibodies were more potent at inducing Bcell lysis than single-chain, bispecific antibodies (BITE®) bearingidentical anti-CD19 and anti-CD3 variable region sequences, with EC₅₀values in the pg/mL range (Moore et al., 2011). Other anti-CD3×anti-CD19bispecific antibodies besides DART® and BITE® have been reported (see,e.g., Wei et al., 2012, Cell Oncol 35:423-34; Portner et al., 2012,Cancer Immunol Immunother 61:1869-75; Zhou et al., 2012, BiotechnolLett. 34:1183-91). In certain embodiments, any known anti-CD3×anti-CD19bispecific antibody may be used to induce an immune response againstdisease-associated cells or pathogens.

In a most preferred embodiment, the anti-CD3×anti-CD19 bispecificantibody is made as a DNL™ construct, as disclosed in Example 1 below.The person of ordinary skill will realize that the subject T-cellredirecting bispecific antibodies are not limited to anti-CD3×anti-CD19constructs, but may comprise antibodies against any knowndisease-associated antigens attached to an anti-CD3 antibody moiety.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. The use ofantibody components derived from humanized, chimeric or human antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Generally, those human FRamino acid residues that differ from their murine counterparts and arelocated close to or touching one or more CDR amino acid residues wouldbe candidates for substitution.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: PHAGE DISPLAY LABORATORY MANUAL, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art (see, e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162).

Phage display can be performed in a variety of formats, for theirreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275,incorporated herein by reference in their entirety. The skilled artisanwill realize that these techniques are exemplary and any known methodfor making and screening human antibodies or antibody fragments may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23)from Abgenix (Fremont, Calif.). In the XENOMOUSE® and similar animals,the mouse antibody genes have been inactivated and replaced byfunctional human antibody genes, while the remainder of the mouse immunesystem remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding Vκ (variable light chain) and V_(H) (variable heavychain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of an antibody from a cell thatexpresses a murine antibody can be cloned by PCR amplification andsequenced. To confirm their authenticity, the cloned V_(L) and V_(H)genes can be expressed in cell culture as a chimeric Ab as described byOrlandi et al., (Proc. Natl. Acad. Sci. USA, 86: 3833 (1989)). Based onthe V gene sequences, a humanized antibody can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine antibody by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The Vκ sequence for the antibody may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human antibody. Alternatively, the VK andV_(H) expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. Antibody fragments are antigen binding portions of anantibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, scFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. F(ab)₂ fragments may be generated by papaindigestion of an antibody.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredescribed in U.S. Pat. No. 4,704,692; U.S. Pat. No. 4,946,778; Raag andWhitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9: 132-137(1991).

Techniques for producing single domain antibodies (DABs or VHH) are alsoknown in the art, as disclosed for example in Cossins et al. (2006, ProtExpress Purif 51:253-259), incorporated herein by reference. Singledomain antibodies may be obtained, for example, from camels, alpacas orllamas by standard immunization techniques. (See, e.g., Muyldermans etal., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75,2003; Maass et al., J Immunol Methods 324:13-25, 2007). The VHH may havepotent antigen-binding capacity and can interact with novel epitopesthat are inaccessible to conventional VH-VL pairs. (Muyldermans et al.,2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgGantibodies (HCAbs) (Maass et al., 2007). Alpacas may be immunized withknown antigens, such as TNF-α, and VHHs can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca VHH coding sequences have been identifiedand may be used to construct alpaca VHH phage display libraries, whichcan be used for antibody fragment isolation by standard biopanningtechniques well known in the art (Maass et al., 2007). In certainembodiments, anti-pancreatic cancer VHH antibody fragments may beutilized in the claimed compositions and methods.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647 and references contained therein.Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960);Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS INENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Stickler et al.,2011). It has been reported that G1m1 antibodies contain allotypicsequences that tend to induce an immune response when administered tonon-G1m1 (nG1m1) recipients, such as G1m3 patients (Stickler et al.,2011). Non-G1m1 allotype antibodies are not as immunogenic whenadministered to G1m1 patients (Stickler et al., 2011).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:85) and veltuzumab (SEQ IDNO:86).

Rituximab heavy chain variable region sequence (SEQ ID NO: 85)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVeltuzuniab heavy chain variable region (SEQ ID NO: 86)ASTKGPSVFLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Known Antibodies

Target Antigens and Exemplary Antibodies

In a preferred embodiment, antibodies are used that recognize and/orbind to antigens that are expressed at high levels on target cells andthat are expressed predominantly or exclusively on diseased cells versusnormal tissues. Exemplary antibodies of use for therapy of, for example,cancer include but are not limited to LL1 (anti-CD74), LL2 or RFB4(anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20),obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor),nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7(anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4 orKC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, alsoknown as CD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6), Mu-9(anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein), R1(anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomabtiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20);PAM4 (aka clivatuzumab, anti-mucin) and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730,300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20050271671;20060193865; 20060210475; 20070087001; the Examples section of eachincorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Other useful antigens that may be targeted using the describedconjugates include carbonic anhydrase IX, B7, CCL19, CCL21, CSAp,HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4,alpha-fetoprotein (AFP), VEGF (e.g., bevacizumab, fibronectin splicevariant), ED-B fibronectin (e.g., L19), EGP-1 (TROP-2), EGP-2 (e.g.,17-1A), EGF receptor (ErbB1) (e.g., cetuximab), ErbB2, ErbB3, Factor H,FHL-1, Flt-3, folate receptor, Ga 733, GRO-β, HMGB-1, hypoxia induciblefactor (HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF),IFN-γ, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2,IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, 1P-10, IGF-1R, Ia,HM1.24, gangliosides, HCG, HLA-DR, CD66 antigens, i.e., CD66a-d or acombination thereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophagemigration-inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac,placental growth factor (PLGF), PSA (prostate-specific antigen), PSMA,PD-1 receptor, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin,S100, tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens, tumornecrosis antigens, tumor angiogenesis antigens, TNF-α, TRAIL receptor(R1 and R2), TROP-2, VEGFR, RANTES, T101, as well as cancer stem cellantigens, complement factors C3, C3a, C3b, C5a, C5, and an oncogeneproduct.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies forzimmunotherapy, is Craig and Foon, Blood prepublished online Jan. 15,2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Perris, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33. Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a) muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Type-1 and Type-2 diabetes may be treated using known antibodies againstB-cell antigens, such as CD22 (epratuzumab and hRFB4), CD74(milatuzumab), CD19 (hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see,e.g., Winer et al., 2011, Nature Med 17:610-18).

The pharmaceutical composition of the present invention may be used totreat a subject having a metabolic disease, such amyloidosis, or aneurodegenerative disease, such as Alzheimer's disease. Bapineuzumab isin clinical trials for Alzheimer's disease therapy. Other antibodiesproposed for therapy of Alzheimer's disease include Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,and solanezumab. Infliximab, an anti-TNF-α antibody, has been reportedto reduce amyloid plaques and improve cognition.

In a preferred embodiment, diseases that may be treated using theclaimed compositions and methods include cardiovascular diseases, suchas fibrin clots, atherosclerosis, myocardial ischemia and infarction.Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1) are known and inclinical trials as imaging agents for disclosing said clots andpulmonary emboli, while anti-granulocyte antibodies, such as MN-3,MN-15, anti-NCA95, and anti-CD15 antibodies, can target myocardialinfarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos. 5,487,892;5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL), anti-MIF, and anti-CD74 (e.g., hLL1) antibodies canbe used to target atherosclerotic plaques. Abciximab (anti-glycoproteinIIb/IIIa) has been approved for adjuvant use for prevention ofrestenosis in percutaneous coronary interventions and the treatment ofunstable angina (Waldmann et al., 2000, Hematol 1:394-408). Antibodiesagainst oxidized LDL induced a regression of established atherosclerosisin a mouse model (Ginsberg, 2007, J Am Coll Cardiol 52:2319-21).Anti-ICAM-1 antibody was shown to reduce ischemic cell damage aftercerebral artery occlusion in rats (Zhang et al., 1994, Neurology44:1747-51). Commercially available monoclonal antibodies to leukocyteantigens are represented by: OKT anti-T-cell monoclonal antibodies(available from Ortho Pharmaceutical Company) which bind to normalT-lymphocytes; the monoclonal antibodies produced by the hybridomashaving the ATCC accession numbers HB44, HB55, HB12, HB78 and HB2; G7Ell,W8E7, NKP15 and G022 (Becton Dickinson); NEN9.4 (New England Nuclear);and FMCll (Sera Labs). A description of antibodies against fibrin andplatelet antigens is contained in Knight, Semin. Nucl. Med., 20:52-67(1990).

An example of a most-preferred antibody/antigen pair is LL1, ananti-CD74 MAb (invariant chain, class II-specific chaperone, Ii) (see,e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; the Examples section of eachincorporated herein by reference). The CD74 antigen is highly expressedon B-cell lymphomas (including multiple myeloma) and leukemias, certainT-cell lymphomas, melanomas, colonic, lung, and renal cancers,glioblastomas, and certain other cancers (Ong et al., Immunology98:296-302 (1999)). A review of the use of CD74 antibodies in cancer iscontained in Stein et al., Clin Cancer Res. 2007 Sep. 15; 13(18 Pt2):5556s-5563s, incorporated herein by reference. The diseases that arepreferably treated with anti-CD74 antibodies include, but are notlimited to, non-Hodgkin's lymphoma, Hodgkin's disease, melanoma, lung,renal, colonic cancers, glioblastome multiforme, histiocytomas, myeloidleukemias, and multiple myeloma.

In another preferred embodiment, the therapeutic conjugates can be usedagainst pathogens, since antibodies against pathogens are known. Forexample, antibodies and antibody fragments which specifically bindmarkers produced by or associated with infectious lesions, includingviral, bacterial, fungal and parasitic infections, for example caused bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi, andviruses, and antigens and products associated with such microorganismshave been disclosed, inter alia, in Hansen et al., U.S. Pat. No.3,927,193 and Goldenberg U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544,4,468,457, 4,444,744, 4,818,709 and 4,624,846, the Examples section ofeach incorporated herein by reference, and in Reichert and Dewitz, citedabove. A review listing antibodies against infectious organisms(antitoxin and antiviral antibodies), as well as other targets, iscontained in Casadevall, Clin Immunol 1999; 93(1):5-15, incorporatedherein by reference.

In a preferred embodiment, the pathogens are selected from the groupconsisting of HIV virus, Mycobacterium tuberculosis, Streptococcusagalactiae, methicillin-resistant Staphylococcus aureus, Legionellapneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcusneoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus,cytomegalovirus, herpes simplex virus I, herpes simplex virus II, humanserum parvo-like virus, respiratory syncytial virus, varicella-zostervirus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus,human T-cell leukemia viruses, Epstein-Barr virus, murine leukemiavirus, mumps virus, vesicular stomatitis virus, sindbis virus,lymphocytic choriomeningitis virus, wart virus, blue tongue virus,Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus40, mouse mammary tumor virus, dengue virus, rubella virus, West Nilevirus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesiabovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica,Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus, Mesocestoides corti,Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae, as disclosed inU.S. Pat. No. 6,440,416, the Examples section of which is incorporatedherein by reference.

Antibodies of use to treat autoimmune disease or immune systemdysfunctions (e.g., graft-versus-host disease, organ transplantrejection) are known in the art and may be used in the disclosed methodsand compositions. Antibodies of use to treat autoimmune/immunedysfunction disease may bind to exemplary antigens including, but notlimited to, BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4, CD5, CD7, CD8,CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD21, CD22,CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43, CD45, CD55,TNF-alpha, interferon and HLA-DR. Antibodies that bind to these andother target antigens, discussed above, may be used to treat autoimmuneor immune dysfunction diseases. Autoimmune diseases that may be treatedwith bsAbs may include acute idiopathic thrombocytopenic purpura,chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham'schorea, myasthenia gravis, systemic lupus erythematosus, lupusnephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid,diabetes mellitus, Henoch-Schonlein purpura, post-streptococcalnephritis, erythema nodosum, Takayasu's arteritis, ANCA-associatedvasculitides, Addison's disease, rheumatoid arthritis, multiplesclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgAnephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture'ssyndrome, thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis, bullouspemphigoid, pemphigus vulgaris, Wegener's granulomatosis, membranousnephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. Antibodies of use maybe commercially obtained from a number of known sources. For example, avariety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets, including but not limitedto tumor-associated antigens, have been deposited at the ATCC and/orhave published variable region sequences and are available for use inthe claimed methods and compositions. See, e.g., U.S. Pat. Nos.7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;6,444,206′ 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953, 5,525,338, the Examples section of eachof which is incorporated herein by reference. These are exemplary onlyand a wide variety of other antibodies and their hybridomas are known inthe art. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

The complement system is a complex cascade involving proteolyticcleavage of serum glycoproteins often activated by cell receptors. The“complement cascade” is constitutive and non-specific but it must beactivated in order to function. Complement activation results in aunidirectional sequence of enzymatic and biochemical reactions. In thiscascade, a specific complement protein, C5, forms two highly active,inflammatory byproducts, C5a and C5b, which jointly activate white bloodcells. This in turn evokes a number of other inflammatory byproducts,including injurious cytokines, inflammatory enzymes, and cell adhesionmolecules. Together, these byproducts can lead to the destruction oftissue seen in many inflammatory diseases. This cascade ultimatelyresults in induction of the inflammatory response, phagocyte chemotaxisand opsonization, and cell lysis.

The complement system can be activated via two distinct pathways, theclassical pathway and the alternate pathway. Most of the complementcomponents are numbered (e.g., C1, C2, C3, etc.) but some are referredto as “Factors.” Some of the components must be enzymatically cleaved toactivate their function; others simply combine to form complexes thatare active. Active components of the classical pathway include C1q, C1r,C1s, C2a, C2b, C3a, C3b, C4a, and C4b. Active components of thealternate pathway include C3a, C3b, Factor B, Factor Ba, Factor Bb,Factor D, and Properdin. The last stage of each pathway is the same, andinvolves component assembly into a membrane attack complex. Activecomponents of the membrane attack complex include C5a, C5b, C6, C7, C8,and C9n.

While any of these components of the complement system can be targetedby an antibody complex, certain of the complement components arepreferred. C3a, C4a and C5a cause mast cells to release chemotacticfactors such as histamine and serotonin, which attract phagocytes,antibodies and complement, etc. These form one group of preferredtargets. Another group of preferred targets includes C3b, C4b and C5b,which enhance phagocytosis of foreign cells. Another preferred group oftargets are the predecessor components for these two groups, i.e., C3,C4 and C5. C5b, C6, C7, C8 and C9 induce lysis of foreign cells(membrane attack complex) and form yet another preferred group oftargets.

Complement C5a, like C3a, is an anaphylatoxin. It mediates inflammationand is a chemotactic attractant for induction of neutrophilic release ofantimicrobial proteases and oxygen radicals. Therefore, C5a and itspredecessor C5 are particularly preferred targets. By targeting C5, notonly is C5a affected, but also C5b, which initiates assembly of themembrane-attack complex. Thus, C5 is another preferred target. C3b, andits predecessor C3, also are preferred targets, as both the classicaland alternate complement pathways depend upon C3b. Three proteins affectthe levels of this factor, C1 inhibitor, protein H and Factor I, andthese are also preferred targets according to the invention. Complementregulatory proteins, such as CD46, CD55, and CD59, may be targets towhich the antibody complexes bind.

Coagulation factors also are preferred targets, particularly tissuefactor and thrombin. Tissue factor is also known also as tissuethromboplastin, CD142, coagulation factor III, or factor III. Tissuefactor is an integral membrane receptor glycoprotein and a member of thecytokine receptor superfamily. The ligand binding extracellular domainof tissue factor consists of two structural modules with features thatare consistent with the classification of tissue factor as a member oftype-2 cytokine receptors. Tissue factor is involved in the bloodcoagulation protease cascade and initiates both the extrinsic andintrinsic blood coagulation cascades by forming high affinity complexesbetween the extracellular domain of tissue factor and the circulatingblood coagulation factors, serine proteases factor VII or factor VIIa.These enzymatically active complexes then activate factor IX and factorX, leading to thrombin generation and clot formation.

Tissue factor is expressed by various cell types, including monocytes,macrophages and vascular endothelial cells, and is induced by IL-1,TNF-α or bacterial lipopolysaccharides. Protein kinase C is involved incytokine activation of endothelial cell tissue factor expression.Induction of tissue factor by endotoxin and cytokines is an importantmechanism for initiation of disseminated intravascular coagulation seenin patients with Gram-negative sepsis. Tissue factor also appears to beinvolved in a variety of non-hemostatic functions includinginflammation, cancer, brain function, immune response, andtumor-associated angiogenesis. Thus, antibody complexes that targettissue factor are useful not only in the treatment of coagulopathies,but also in the treatment of sepsis, cancer, pathologic angiogenesis,and other immune and inflammatory dysregulatory diseases according tothe invention. A complex interaction between the coagulation pathway andthe cytokine network is suggested by the ability of several cytokines toinfluence tissue factor expression in a variety of cells and by theeffects of ligand binding to the receptor. Ligand binding (factor VIIa)has been reported to give an intracellular calcium signal, thusindicating that tissue factor is a true receptor.

Thrombin is the activated form of coagulation factor II (prothrombin);it converts fibrinogen to fibrin. Thrombin is a potent chemotaxin formacrophages, and can alter their production of cytokines and arachidonicacid metabolites. It is of particular importance in the coagulopathiesthat accompany sepsis. Numerous studies have documented the activationof the coagulation system either in septic patients or following LPSadministration in animal models. Despite more than thirty years ofresearch, the mechanisms of LPS-induced liver toxicity remain poorlyunderstood. It is now clear that they involve a complex and sequentialseries of interactions between cellular and humoral mediators. In thesame period of time, gram-negative systemic sepsis and its sequalae havebecome a major health concern, attempts to use monoclonal antibodiesdirected against LPS or various inflammatory mediators have yielded onlytherapeutic failures. antibody complexes that target both thrombin andat least one other target address the clinical failures in sepsistreatment.

In other embodiments, the antibody complexes bind to a MHC class I, MHCclass II or accessory molecule, such as CD40, CD54, CD80 or CD86. Theantibody complex also may bind to a T-cell activation cytokine, or to acytokine mediator, such as NF-κB.

In certain embodiments, one of the two different targets may be a cancercell receptor or cancer-associated antigen, particularly one that isselected from the group consisting of B-cell lineage antigens (CD19,CD20, CD21, CD22, CD23, etc.), VEGF, VEGFR, EGFR, carcinoembryonicantigen (CEA), placental growth factor (PlGF), tenascin, HER-2/neu,EGP-1, EGP-2, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD80,CD138, NCA66, CEACAM1, CEACAM6 (carcinoembryonic antigen-relatedcellular adhesion molecule 6), MUC1, MUC2, MUC3, MUC4, MUC16, IL-6,α-fetoprotein (AFP), A3, CA125, colon-specific antigen-p (CSAp), folatereceptor, HLA-DR, human chorionic gonadotropin (HCG), Ia, EL-2,insulin-like growth factor (IGF) and IGF receptor, KS-1, Le(y), MAGE,necrosis antigens, PAM-4, prostatic acid phosphatase (PAP), Pr1,prostate specific antigen (PSA), prostate specific membrane antigen(PSMA), S100, T101, TAC, TAG72, TRAIL receptors, and carbonic anhydraseIX.

Targets associated with sepsis and immune dysregulation and other immunedisorders include MIF, IL-1, IL-6, IL-8, CD74, CD83, and C5aR.Antibodies and inhibitors against C5aR have been found to improvesurvival in rodents with sepsis (Huber-Lang et al., FASEB J 2002;16:1567-1574; Riedemann et al., J Clin Invest 2002; 110:101-108) andseptic shock and adult respiratory distress syndrome in monkeys (Hangenet al., J Surg Res 1989; 46:195-199; Stevens et al., J Clin Invest 1986;77:1812-1816). Thus, for sepsis, one of the two different targetspreferably is a target that is associated with infection, such asLPS/C5a. Other preferred targets include HMGB-1, tissue factor, CD14,VEGF, and IL-6, each of which is associated with septicemia or septicshock. Preferred antibody complexes are those that target two or moretargets from HMGB-1, tissue factor and MIF, such as MIF/tissue factor,and HMGB-1/tissue factor.

In still other embodiments, one of the different targets may be a targetthat is associated with graft versus host disease or transplantrejection, such as MIF (Lo et al., Bone Marrow Transplant, 30(6):375-80(2002)). One of the different targets also may be one that associatedwith acute respiratory distress syndrome, such as IL-8 (Bouros et al.,PMC Pulm Med, 4(1):6 (2004), atherosclerosis or restenosis, such as MIF(Chen et al., Arterioscler Thromb Vasc Biol, 24(4):709-14 (2004),asthma, such as IL-18 (Hata et al., Int Immunol, Oct. 11, 2004 Epubahead of print), a granulomatous disease, such as TNF-α (Ulbricht etal., Arthritis Rheum, 50(8):2717-8 (2004), a neuropathy, such ascarbamylated EPO (erythropoietin) (Leist et al., Science 305(5681):164-5(2004), or cachexia, such as IL-6 and TNF-α.

Other targets include C5a, LPS, IFN-gamma, B7; CD2, CD4, CD14, CD18,CD11a, CD11b, CD11c, CD14, CD18, CD27, CD29, CD38, CD40L, CD52, CD64,CD83, CD147, CD154. Activation of mononuclear cells by certain microbialantigens, including LPS, can be inhibited to some extent by antibodiesto CD18, CD11b, or CD11c, which thus implicate β₂-integrins (Cuzzola etal., J Immunol 2000; 164:5871-5876; Medvedev et al., J Immunol 1998;160: 4535-4542). CD83 has been found to play a role in giant cellarteritis (GCA), which is a systemic vasculitis that affects medium- andlarge-size arteries, predominately the extracranial branches of theaortic arch and of the aorta itself, resulting in vascular stenosis andsubsequent tissue ischemia, and the severe complications of blindness,stroke and aortic arch syndrome (Weyand and Goronzy, N Engl J Med 2003;349:160-169; Hunder and Valente, In: INFLAMMATORY DISEASES OF BLOODVESSELS. G. S. Hoffman and C. M. Weyand, eds, Marcel Dekker, New York,2002; 255-265). Antibodies to CD83 were found to abrogate vasculitis ina SCID mouse model of human GCA (Ma-Krupa et al., J Exp Med 2004;199:173-183), suggesting to these investigators that dendritic cells,which express CD83 when activated, are critical antigen-processing cellsin GCA. In these studies, they used a mouse anti-CD83 MAb (IgG1 cloneHB15e from Research Diagnostics). CD154, a member of the TNF family, isexpressed on the surface of CD4-positive T-lymphocytes, and it has beenreported that a humanized monoclonal antibody to CD154 producedsignificant clinical benefit in patients with active systemic lupuserythematosus (SLE) (Grammar et al., J Clin Invest 2003; 112:1506-1520).It also suggests that this antibody might be useful in other autoimmunediseases (Kelsoe, J Clin Invest 2003; 112:1480-1482). Indeed, thisantibody was also reported as effective in patients with refractoryimmune thrombocytopenic purpura (Kuwana et al., Blood 2004;103:1229-1236).

In rheumatoid arthritis, a recombinant interleukin-1 receptorantagonist, IL-1Ra or anakinra, has shown activity (Cohen et al., AnnRheum Dis 2004; 63:1062-8; Cohen, Rheum Dis Clin North Am 2004;30:365-80). An improvement in treatment of these patients, whichhitherto required concomitant treatment with methotrexate, is to combineanakinra with one or more of the anti-proinflammatory effector cytokinesor anti-proinflammatory effector chemokines (as listed above). Indeed,in a review of antibody therapy for rheumatoid arthritis, Taylor (CurrOpin Pharmacol 2003; 3:323-328) suggests that in addition to TNF, otherantibodies to such cytokines as IL-1, IL-6, IL-8, IL-15, IL-17 andIL-18, are useful.

The pharmaceutical composition of the present invention may be used totreat a subject having a metabolic disease, such amyloidosis, or aneurodegenerative disease, such as Alzheimer's disease. Bapineuzumab isin clinical trials for Alzheimer's disease therapy. Other antibodiesproposed for therapy of Alzheimer's disease include Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,and solanezumab. Infliximab, an anti-TNF-α antibody, has been reportedto reduce amyloid plaques and improve cognition.

In a preferred embodiment, diseases that may be treated using theclaimed compositions and methods include cardiovascular diseases, suchas fibrin clots, atherosclerosis, myocardial ischemia and infarction.Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1) are known and inclinical trials as imaging agents for disclosing said clots andpulmonary emboli, while anti-granulocyte antibodies, such as MN-3,MN-15, anti-NCA95, and anti-CD15 antibodies, can target myocardialinfarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos. 5,487,892;5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL), anti-MIF, and anti-CD74 (e.g., hLL1) antibodies canbe used to target atherosclerotic plaques. Abciximab (anti-glycoproteinIIb/IIIa) has been approved for adjuvant use for prevention ofrestenosis in percutaneous coronary interventions and the treatment ofunstable angina (Waldmann et al., 2000, Hematol 1:394-408). Antibodiesagainst oxidized LDL induced a regression of established atherosclerosisin a mouse model (Ginsberg, 2007, J Am Coll Cardiol 52:2319-21).Anti-ICAM-1 antibody was shown to reduce ischemic cell damage aftercerebral artery occlusion in rats (Zhang et al., 1994, Neurology44:1747-51). Commercially available monoclonal antibodies to leukocyteantigens are represented by: OKT anti-T-cell monoclonal antibodies(available from Ortho Pharmaceutical Company) which bind to normalT-lymphocytes; the monoclonal antibodies produced by the hybridomashaving the ATCC accession numbers HB44, HB55, HB12, HB78 and HB2; G7Ell,W8E7, NKP15 and GO22 (Becton Dickinson); NEN9.4 (New England Nuclear);and FMCll (Sera Labs). A description of antibodies against fibrin andplatelet antigens is contained in Knight, Semin. Nucl. Med., 20:52-67(1990).

Other antibodies that may be used include antibodies against infectiousdisease agents, such as bacteria, viruses, mycoplasms or otherpathogens. Many antibodies against such infectious agents are known inthe art and any such known antibody may be used in the claimed methodsand compositions. For example, antibodies against the gp120 glycoproteinantigen of human immunodeficiency virus I (HIV-1) are known, and certainof such antibodies can have an immunoprotective role in humans. See,e.g., Rossi et al., Proc. Natl. Acad. Sci. USA. 86:8055-8058, 1990.Known anti-HIV antibodies include the anti-envelope antibody describedby Johansson et al. (AIDS, 2006 Oct. 3; 20(15):1911-5), as well as theanti-HIV antibodies described and sold by Polymun (Vienna, Austria),also described in U.S. Pat. No. 5,831,034, U.S. Pat. No. 5,911,989, andVcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob.Agents Chemother. 2006; 50(5):1773-9, all incorporated herein byreference.

Antibodies against malaria parasites can be directed against thesporozoite, merozoite, schizont and gametocyte stages. Monoclonalantibodies have been generated against sporozoites (cirumsporozoiteantigen), and have been shown to neutralize sporozoites in vitro and inrodents (N. Yoshida et al., Science 207:71-73, 1980). Several groupshave developed antibodies to T. gondii, the protozoan parasite involvedin toxoplasmosis (Kasper et al., J. Immunol. 129:1694-1699, 1982; Id.,30:2407-2412, 1983). Antibodies have been developed againstschistosomular surface antigens and have been found to act againstschistosomulae in vivo or in vitro (Simpson et al., Parasitology,83:163-177, 1981; Smith et al., Parasitology, 84:83-91, 1982: Gryzch etal., J. Immunol., 129:2739-2743, 1982; Zodda et al., J. Immunol.129:2326-2328, 1982; Dissous et al., J. Immunol., 129:2232-2234, 1982)

Trypanosoma cruzi is the causative agent of Chagas' disease, and istransmitted by blood-sucking reduviid insects. An antibody has beengenerated that specifically inhibits the differentiation of one form ofthe parasite to another (epimastigote to trypomastigote stage) in vitro,and which reacts with a cell-surface glycoprotein; however, this antigenis absent from the mammalian (bloodstream) forms of the parasite (Sheret al., Nature, 300:639-640, 1982).

Anti-fungal antibodies are known in the art, such as anti-Sclerotiniaantibody (U.S. Pat. No. 7,910,702); antiglucuronoxylomannan antibody(Zhong and Priofski, 1998, Clin Diag Lab Immunol 5:58-64); anti-Candidaantibodies (Matthews and Burnie, 2001, Curr Opin Investig Drugs2:472-76); and anti-glycosphingolipid antibodies (Toledo et al., 2010,BMC Microbiol 10:47).

Suitable antibodies have been developed against most of themicroorganism (bacteria, viruses, protozoa, fungi, other parasites)responsible for the majority of infections in humans, and many have beenused previously for in vitro diagnostic purposes. These antibodies, andnewer antibodies that can be generated by conventional methods, areappropriate for use in the present invention.

Immunoconjugates

In certain embodiments, the antibodies or fragments thereof may beconjugated to one or more therapeutic or diagnostic agents. Thetherapeutic agents do not need to be the same but can be different, e.g.a drug and a radioisotope. For example, ¹³¹I can be incorporated into atyrosine of an antibody or fusion protein and a drug attached to anepsilon amino group of a lysine residue. Therapeutic and diagnosticagents also can be attached, for example to reduced SH groups and/or tocarbohydrate side chains. Many methods for making covalent ornon-covalent conjugates of therapeutic or diagnostic agents withantibodies or fusion proteins are known in the art and any such knownmethod may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein and used to chelate a therapeutic ordiagnostic agent, such as a radionuclide. Exemplary chelators includebut are not limited to DIVA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.Methods of conjugation and use of chelating agents to attach metals orother ligands to proteins are well known in the art (see, e.g., U.S.Pat. No. 7,563,433, the Examples section of which is incorporated hereinby reference).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein in itsentirety by reference. Particularly useful metal-chelate combinationsinclude 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, usedwith diagnostic isotopes in the general energy range of 60 to 4,000 keV,such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga,^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radioimaging. The samechelates, when complexed with non-radioactive metals, such as manganese,iron and gadolinium are useful for MRI. Macrocyclic chelates such asNOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F—Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. Pat. No.7,563,433, the Examples section of which is incorporated herein byreference.

DOCK-AND-LOCK™ (DNL™)

In preferred embodiments, a bispecific antibody, either alone or elsecomplexed to one or more effectors such as cytokines, is formed as aDOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,901,680; 7,906,118;7,981,398; 8,003,111, the Examples section of each of which isincorporated herein by reference.) Generally, the technique takesadvantage of the specific and high-affinity binding interactions thatoccur between a dimerization and docking domain (DDD) sequence of theregulatory (R) subunits of cAMP-dependent protein kinase (PKA) and ananchor domain (AD) sequence derived from any of a variety of AKAPproteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and AD peptides may beattached to any protein, peptide or other molecule. Because the DDDsequences spontaneously dimerize and bind to the AD sequence, thetechnique allows the formation of complexes between any selectedmolecules that may be attached to DDD or AD sequences.

Although the standard DNL™ complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL™complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL™ complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ, each of which comprises a DDD moiety amino acid sequence. The Rsubunits have been isolated only as stable dimers and the dimerizationdomain has been shown to consist of the first 44 amino-terminal residuesof RIIα (Newlon et al., Nat. Struct. Biol. 1999; 6:222). As discussedbelow, similar portions of the amino acid sequences of other regulatorysubunits are involved in dimerization and docking, each located near theN-terminal end of the regulatory subunit. Binding of cAMP to the Rsubunits leads to the release of active catalytic subunits for a broadspectrum of serine/threonine kinase activities, which are orientedtoward selected substrates through the compartmentalization of PKA viaits docking with AKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are varied amongindividual AKAPs, with the binding affinities reported for RII dimersranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA 2003;100:4445). AKAPs will only bind to dimeric R subunits. For human RIIα,the AD binds to a hydrophobic surface formed by the 23 amino-terminalresidues (Colledge and Scott, Trends Cell Biol. 1999; 6:216). Thus, thedimerization domain and AKAP binding domain of human RIIα are bothlocated within the same N-terminal 44 amino acid sequence (Newlon etal., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J. 2001;20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL™complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₁b. This binding event is stabilized with a subsequentreaction to covalently secure the two entities via disulfide bridges,which occurs very efficiently based on the principle of effective localconcentration because the initial binding interactions should bring thereactive thiol groups placed onto both the DDD and AD into proximity(Chmura et al., Proc. Natl. Acad. Sci. USA 2001; 98:8480) to ligatesite-specifically. Using various combinations of linkers, adaptormodules and precursors, a wide variety of DNL™ constructs of differentstoichiometry may be produced and used (see, e.g., U.S. Pat. Nos.7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL™construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Dock-and-Lock™ (DNL™) technology has been used to produce a variety ofcomplexes in assorted formats (Rossi et al., 2012, Bioconjug Chem23:309-23). Bispecific hexavalent antibodies (bsHexAbs) based onveltuzumab (anti-CD20) and epratuzumab (anti-CD22) were constructed bycombining a stabilized (Fab)₂ fused to a dimerization and docking domain(DDD) with an IgG containing an anchor domain (AD) appended at theC-terminus of each heavy chain (C_(H)3-AD2-IgG) (Rossi et al., 2009,Blood 113, 6161-71). Compared to mixtures of their parental mAbs, theseFc-based bsHexAbs, referred to henceforth as “Fc-bsHexAbs”, inducedunique signaling events (Gupta et al., 2010, Blood 116:3258-67), andexhibited potent cytotoxicity in vitro. However, the Fc-bsHexAbs werecleared from circulation of mice approximately twice as fast as theparental mAbs (Rossi et al., 2009, Blood 113, 6161-71). Although theFc-bsHexAbs are highly stable ex vivo, it is possible that somedissociation occurs in vivo, for example by intracellular processing.Further, the Fc-bsHexAbs lack CDC activity.

Fc-based immunocytokines have also been assembled as DNL™ complexes,comprising two or four molecules of interferon-alpha 2b (IFNα2b) fusedto the C-terminal end of the C_(H)3-AD2-IgG Fc (Rossi et al., 2009,Blood 114:3864-71; Rossi et al., 2010, Cancer Res 70:7600-09; Rossi etal., 2011, Blood 118:1877-84). The Fc-IgG-IFNα maintained high specificactivity, approaching that of recombinant IFNα, and were remarkablypotent in vitro and in vivo against non-Hodgkin lymphoma (NHL)xenografts. The T_(1/2) of the Fc-IgG-IFNα in mice was longer thanPEGylated IFNα, but half as long as the parental mAbs. Similar to theFc-bsHexAbs, the Fc-IgG-IFNα dissociated in vivo over time and exhibiteddiminished CDC, but ADCC was enhanced.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL™ constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL™ complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 9)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEEN RQILA PKA RIIα(SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 2  Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 87. S H I Q I P P G L T E L LQ G Y T V E V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F TR L R E A R A N N E D L D S K K D L K L I I I V V V

(SEQ ID NO: 12) THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 13) SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 14) SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 15) SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 16) SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 17) SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 18) SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 19) SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 20) SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 21) SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFIRLREARA(SEQ ID NO: 22) SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 23) SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 24) SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 25) SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA(SEQ ID NO: 26) SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA(SEQ ID NO: 27) SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA(SEQ ID NO: 28) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA(SEQ ID NO: 29) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA(SEQ ID NO: 30) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA(SEQ ID NO: 31) SHIQEPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 2 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

TABLE 3  Conservative Amino Acid Substitutions in AD1 (SEQ ID NO: 3).Consensus sequence disclosed as SEQ ID NO: 88. Q I E Y L A K Q I V D N AI Q Q A N L D F I R N E Q N N L V T V I S V

(SEQ ID NO: 32) NIEYLAKQIVDNAIQQA (SEQ ID NO: 33) QLEYLAKQIVDNAIQQA(SEQ ID NO: 34) QVEYLAKQIVDNAIQQA (SEQ ID NO: 35) QIDYLAKQIVDNAIQQA(SEQ ID NO: 36) QIEFLAKQIVDNAIQQA (SEQ ID NO: 37) QIETLAKQIVDNAIQQA(SEQ ID NO: 38) QIESLAKQIVDNAIQQA (SEQ ID NO: 39) QIEYIAKQIVDNAIQQA(SEQ ID NO: 40) QIEYVAKQIVDNAIQQA (SEQ ID NO: 41) QIEYLARQIVDNAIQQA(SEQ ID NO: 42) QIEYLAKNIVDNAIQQA (SEQ ID NO: 43) QIEYLAKQIVENAIQQA(SEQ ID NO: 44) QIEYLAKQIVDQAIQQA (SEQ ID NO: 45) QIEYLAKQIVDNAINQA(SEQ ID NO: 46) QIEYLAKQIVDNAIQNA (SEQ ID NO: 47) QIEYLAKQIVDNAIQQL(SEQ ID NO: 48) QIEYLAKQIVDNAIQQI (SEQ ID NO: 49) QIEYLAKQIVDNAIQQV

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL™ constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA(SEQ ID NO: 52) QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL (SEQ ID NO: 54) PLEYQAGLLVQNAIQQAI AKAP79(SEQ ID NO: 55) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 56)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 57)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 58) LEQVANQLADQIIKEAT PV38(SEQ ID NO: 59) FEELAWKIAKMIWSDVF Dual-Specificity AKAPs AKAP7(SEQ ID NO: 60) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 61)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 62) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 63) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RH isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 65)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4  AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-PQIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pepETSAKDNINIHEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5  Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO:1).Consensus sequence disclosed as SEQ ID NO:89. S H I Q

P

T E

Q

V

T N S I L A Q

P

V E

V E

T R

R E A

A N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL™ constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−.0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).Replacement of amino acids with others of similar hydrophilicity ispreferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, vat; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (q) glu, asn; Glu (E) gln, asp; Gly (G) ala; H is (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Therapeutic Agents

In alternative embodiments, therapeutic agents such as cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones,hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes orother agents may be used, either conjugated to the subject bsAbs orseparately administered before, simultaneously with, or after the bsAb.Drugs of use may possess a pharmaceutical property selected from thegroup consisting of antimitotic, antikinase, alkylating, antimetabolite,antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents andcombinations thereof.

Exemplary drugs of use may include, but are not limited to,5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptorbinding agents, etoposide (VP16), etoposide glucuronide, etoposidephosphate, exemestane, fingolimod, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib,ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea,ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase,lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PlGFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -λ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT.

Radionuclides of use include, but are not limited to—¹¹¹In, ¹⁷¹Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb, and ²²⁷Th. The therapeuticradionuclide preferably has a decay-energy in the range of 20 to 6,000keV, preferably in the ranges 60 to 200 keV for an Auger emitter,100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alphaemitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255. Decayenergies of useful alpha-particle-emitting radionuclides are preferably2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably4,000-7,000 keV. Additional potential radioisotopes of use include ¹¹C,¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru,¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm,¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au,⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.Some useful diagnostic nuclides may include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, or ¹¹¹In.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bcl-2 or p53. Apreferred form of therapeutic oligonucleotide is siRNA. The skilledartisan will realize that any siRNA or interference RNA species may beattached to an antibody or fragment thereof for delivery to a targetedtissue. Many siRNA species against a wide variety of targets are knownin the art, and any such known siRNA may be utilized in the claimedmethods and compositions.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), andapolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of eachreferenced patent incorporated herein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Mirus Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either a siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Any such siRNA species may be delivered usingthe subject DNL complexes.

Methods of Therapeutic Treatment

Various embodiments concern methods of treating a cancer in a subject,such as a mammal, including humans, domestic or companion pets, such asdogs and cats, comprising administering to the subject a therapeuticallyeffective amount of a cytotoxic bsAb.

In one embodiment, immunological diseases which may be treated with thesubject bsAbs may include, for example, joint diseases such asankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoidarthritis; neurological disease such as multiple sclerosis andmyasthenia gravis; pancreatic disease such as diabetes, especiallyjuvenile onset diabetes; gastrointestinal tract disease such as chronicactive hepatitis, celiac disease, ulcerative colitis, Crohn's disease,pernicious anemia; skin diseases such as psoriasis or scleroderma;allergic diseases such as asthma and in transplantation relatedconditions such as graft versus host disease and allograft rejection.

The administration of the cytotoxic bsAbs can be supplemented byadministering concurrently or sequentially a therapeutically effectiveamount of another antibody that binds to or is reactive with anotherantigen on the surface of the target cell. Preferred additional MAbscomprise at least one humanized, chimeric or human MAb selected from thegroup consisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD16,CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD30, CD32b, CD33, CD37,CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD70, CD74, CD79a, CD80,CD95, CD126, CD133, CD138, CD154, CEACAM5, CEACAM6, B7, AFP, PSMA,EGP-1, EGP-2, carbonic anhydrase IX, PAM4 antigen, MUC1, MUC2, MUC3,MUC4, MUC5, Ia, MIF, HM1.24, HLA-DR, tenascin, Flt-3, VEGFR, PlGF, ILGF,IL-6, IL-25, tenascin, TRAIL-R1, TRAIL-R2, complement factor C5,oncogene product, or a combination thereof. Various antibodies of use,such as anti-CD19, anti-CD20, and anti-CD22 antibodies, are known tothose of skill in the art. See, for example, Ghetie et al., Cancer Res.48:2610 (1988); Heiman et al., Cancer Immunol. Immunother. 32:364(1991); Longo, Curr. Opin. Oncol. 8:353 (1996), U.S. Pat. Nos.5,798,554; 6,187,287; 6,306,393; 6,676,924; 7,109,304; 7,151,164;7,230,084; 7,230,085; 7,238,785; 7,238,786; 7,282,567; 7,300,655;7,312,318; 7,501,498; 7,612,180; 7,670,804; and U.S. Patent ApplicationPubl. Nos. 20080131363; 20070172920; 20060193865; and 20080138333, theExamples section of each incorporated herein by reference.

The bsAb therapy can be further supplemented with the administration,either concurrently or sequentially, of at least one therapeutic agent.For example, “CVB” (1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide,and 150-200 mg/m² carmustine) is a regimen used to treat non-Hodgkin'slymphoma. Patti et al., Eur. J. Haematol. 51: 18 (1993). Other suitablecombination chemotherapeutic regimens are well-known to those of skillin the art. See, for example, Freedman et al., “Non-Hodgkin'sLymphomas,” in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al.(eds.), pages 2028-2068 (Lea & Febiger 1993). As an illustration, firstgeneration chemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide,vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide,doxorubicin, vincristine, and prednisone). A useful second generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate, bendamustine, and bryostatin-1.

The subject bsAbs can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby the bsAb iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

The subject bsAbs can be formulated for intravenous administration via,for example, bolus injection or continuous infusion. Preferably, thebsAb is infused over a period of less than about 4 hours, and morepreferably, over a period of less than about 3 hours. For example, thefirst bolus could be infused within 30 minutes, preferably even 15 min,and the remainder infused over the next 2-3 hrs. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the bsAbs. Control release preparations can beprepared through the use of polymers to complex or adsorb the bsAbs. Forexample, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release from such a matrixdepends upon the molecular weight of the bsAb, the amount of bsAb withinthe matrix, and the size of dispersed particles. Saltzman et al.,Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid dosageforms are described in Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

The bsAb may also be administered to a mammal subcutaneously or even byother parenteral routes, such as intravenously, intramuscularly,intraperitoneally or intravascularly. Moreover, the administration maybe by continuous infusion or by single or multiple boluses. Preferably,the bsAb is infused over a period of less than about 4 hours, and morepreferably, over a period of less than about 3 hours.

More generally, the dosage of an administered bsAb for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. It may bedesirable to provide the recipient with a dosage of bsAb that is in therange of from about 1 mg/kg to 25 mg/kg as a single intravenousinfusion, although a lower or higher dosage also may be administered ascircumstances dictate. A dosage of 1-20 mg/kg for a 70 kg patient, forexample, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosagemay be repeated as needed, for example, once per week for 4-10 weeks,once per week for 8 weeks, or once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months, ormonthly or quarterly for many months, as needed in a maintenancetherapy.

Alternatively, a bsAb may be administered as one dosage every 2 or 3weeks, repeated for a total of at least 3 dosages. Or, the construct maybe administered twice per week for 4-6 weeks. If the dosage is loweredto approximately 200-300 mg/m² (340 mg per dosage for a 1.7-m patient,or 4.9 mg/kg for a 70 kg patient), it may be administered once or eventwice weekly for 4 to 10 weeks. Alternatively, the dosage schedule maybe decreased, namely every 2 or 3 weeks for 2-3 months. It has beendetermined, however, that even higher doses, such as 20 mg/kg onceweekly or once every 2-3 weeks can be administered by slow i.v.infusion, for repeated dosing cycles. The dosing schedule can optionallybe repeated at other intervals and dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

While the bsAbs may be administered as a periodic bolus injection, inalternative embodiments the bsAbs may be administered by continuousinfusion. In order to increase the Cmax and extend the PK of the bsAbsin the blood, a continuous infusion may be administered for example byindwelling catheter. Such devices are known in the art, such asHICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see, e.g., Skolnik et al.,Ther Drug Monit 32:741-48, 2010) and any such known indwelling cathetermay be used. A variety of continuous infusion pumps are also known inthe art and any such known infusion pump may be used. The dosage rangefor continuous infusion may be between 0.1 and 3.0 mg/kg per day. Morepreferably, the bsAbs can be administered by intravenous infusions overrelatively short periods of 2 to 5 hours, more preferably 2-3 hours.

In preferred embodiments, the bsAbs are of use for therapy of cancer.Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastomamultiforme, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors,medullary thyroid cancer, differentiated thyroid carcinoma, breastcancer, ovarian cancer, colon cancer, rectal cancer, endometrial canceror uterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, aswell as head-and-neck cancer. The term “cancer” includes primarymalignant cells or tumors (e.g., those whose cells have not migrated tosites in the subject's body other than the site of the originalmalignancy or tumor) and secondary malignant cells or tumors (e.g.,those arising from metastasis, the migration of malignant cells or tumorcells to secondary sites that are different from the site of theoriginal tumor). Cancers conducive to treatment methods of the presentinvention involves cells which express, over-express, or abnormallyexpress IGF-1R.

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational TROPhoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, TROPhoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BASICPATHOLOGY, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding an antibody, antibody fragment, cytokine or constituentfusion protein of a bsAb, such as a DNL™ construct. Fusion proteins maycomprise an antibody or fragment or cytokine attached to, for example,an AD or DDD moiety.

Various embodiments relate to expression vectors comprising the codingDNA sequences. The vectors may contain sequences encoding the light andheavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express the encoded protein(s) in a selected host cell,enhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine. See Edelmanet al., Proc. Natl. Acad. Sci. USA 63: 78-85 (1969). In otherembodiments, an IgG1 sequence may be converted to an IgG4 sequence.

The skilled artisan will realize that methods of genetically engineeringexpression constructs and insertion into host cells to expressengineered proteins are well known in the art and a matter of routineexperimentation. Host cells and methods of expression of clonedantibodies or fragments have been described, for example, in U.S. Pat.Nos. 7,531,327, 7,537,930, 7,785,880, 8,076,410, 8,153,433 and8,372,603, the Examples section of each incorporated herein byreference.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain one or more bsAbs as described herein. If the compositioncontaining components for administration is not formulated for deliveryvia the alimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included.One type of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used. In certain embodiments, atherapeutic agent may be provided in the form of a prefilled syringe orautoinjection pen containing a sterile, liquid formulation orlyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

The following examples are provided to illustrate, but not to limit, theclaims of the present invention.

Example 1 T-Cell Redirecting Bispecific Antibody DOCK-AND-LOCK™ (DNL™)Complexes

Several species of exemplary T-cell redirecting bispecific antibodieswere made as DNL™ complexes, as described below. The complexes wereeffective to induce an immune response against appropriate target cells.

Materials and Methods

General techniques for making and using DOCK-AND-LOCK™ (DNL™) complexesare described in the Examples below. An exemplary T-cell redirectingbispecific antibody with binding sites for CD3 and CD19 was made as aDNL™ complex, referred to as (19)-3s (FIG. 1). An anti-CD19 F(ab)₂ DNLmodule was constructed by recombinant fusion of a dimerization anddocking domain (DDD2) at the carboxyl terminal end of the Fd chain. Ananti-CD3-scFv module was designed from Okt3 mAb with addition of ananchor domain (AD2) and assembled in the formatV_(H)-L1-V_(K)-L2-6H-L3-AD2 (“6H” disclosed as SEQ ID NO:105), where theV domains were fused via a flexible peptide linker and the AD2 peptidewas preceded by a 6-His linker (SE ID NO:105). The sequences of theanti-CD3 variable regions, linkers and AD2 were as shown below.

V_(H) sequence of anti-CD3 scFv (SEQ ID NO: 96)QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTT LTVSSL1 Linker (SEQ ID NO: 97) GGGGSGGGGSGGGGSV_(K) sequence of anti-CD3 scFv (SEQ ID NO: 98)DIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIKR L2 Linker(SEQ ID NO: 99) GGGGS Poly-His-L3 Linker (SEQ ID NO: 100) HHHHHHGGGSGAD2 (SEQ ID NO: 101) CGQIEYLAKQIVDNAIQQAGC

Expression Vectors and DNL™ Modules—

DNL™ complexes were constructed comprising antibody moieties againstvarious disease-associated antigens, linked to an anti-CD3 antibodymoiety, generally abbreviated as (X)-3s bsAbs. Independent productioncell lines were developed in SpESFX-10 mouse myeloma cells (Rossi etal., 2011, Biotechnol Prog 27:766-75) for each of the DNL™ modules usedto make the (X)-3s bsAbs. A cDNA sequence encoding the Okt3scFv-AD2polypeptide (SEQ ID NOs:96-101) was synthesized and cloned into thepdHL2 expression vector via 5′ Xba I and 3′ Eag I restriction sites. Theconstruct comprised the V_(H) domain fused to the V_(L) in an scFv withthe structure V_(H)-L1-V_(K)-L2-6H-L3-AD2 (“6H” disclosed as SEQ IDNO:105). The expressed protein had two amino acid substitutions from theoriginal Okt3 mAb. A cysteine residue in the CDR-H3 was changed toserine (Kipryanov, 1997, J Immunol Methods 200:69-77). The penultimateresidue of the V_(L) was changed from aspartate to lysine.

The Okt3scFv-AD2 module was combined with various C_(H)1-DDD2-Fabmodules to generate a panel of (X)-3s trivalent bsAbs (Table 6). TheC_(H)1-DDD2-Fab-pdHL2 expression vectors were constructed as describedpreviously for similar constructs (Rossi et al., 2008, Cancer Res68:8384-92). Briefly, expression vectors encoding C_(H)1-DDD2-Fab weregenerated from the corresponding IgG-pdHL2 expression vectors byexcising the coding sequence for the C_(H)1-Hinge-C_(H)2-C_(H)3 domainswith Sac II and Eag I restriction enzymes and replacing it with a 507 bpsequence encoding C_(H)1-DDD2, which was excised from theC_(H)1-DDD2-Fab-hA20-pdHL2 expression vector (Rossi et al., 2008, CancerRes 68:8384-92) with the same enzymes. C_(H)1-DDD2-Fab modules werederived from the humanized mAbs hA19 (anti-CD19), labetuzumab (hMN-14,anti-CEACAM5), clivatuzumab (hPAM4, anti-mucin), hMN-15 (anti-CEACAM6),hRS7 (anti-TROP-2), veltuzumab (hA20, anti-CD20), hL243 (anti-HLA-DR)and epratuzumab (hLL2, anti-CD22). The mAb designated hA19 was humanizedfrom the mouse anti-CD19 mAb B43 (Uckun et al., 1988, Blood 71:13-29).Each expression vector was linearized by digestion with Sal Irestriction enzyme and used to transfect SpESFX-10 cells byelectroporation.

Clones were selected in media containing 0.2 μM methotrexate (MTX) andscreened for protein expression by ELISA. Okt3scFv-AD2 was captured onNi-NTA H isSorb plates (Qiagen) and detected with an anti-AD2 mAb.C_(H)1-DDD2-Fab modules were captured with goat-anti-human-kappa chainand detected with goat-anti-human-F(ab′)₂-HRP. Productivity ofprotein-expression was amplified by stepwise increases in MTXconcentration up to 3 μM. Okt3scFv-AD2 and C_(H)1-DDD2-Fab modules werepurified to homogeneity from the broth of roller bottle cultures byaffinity chromatography using Ni-SEPHAROSE® and Kappa-Select resins,respectively. The DNL™ method was used to assemble (X)-3s bsAbs via thesite-specific conjugation of mole equivalents of Okt3scFv-AD2 andC_(H)1-DDD2-Fab modules. For example, approximately 100 mg of (19)-3swere produced by combining 22 mg of Okt3scFv-AD2 with 80 mg ofC_(H)1-DDD2-Fab-hA19. The mixture was reduced overnight at roomtemperature with 1 mM reduced glutathione prior to the addition of 2 mMoxidized glutathione. The (19)-3s was purified from the reaction mixtureby sequential affinity chromatography with Kappa-Select andNi-SEPHAROSE®. Additional (X)-3s constructs were assembled at variousscales following a similar process.

TABLE 6 (X)-3s DNL ™ Constructs Code Target C_(H)1-DDD2-Fab AD2-anti-CD3(19)-3s CD19 C_(H)1-DDD2-Fab-hA19 scFv-AD2- Okt3 (20)-3s CD20C_(H)1-DDD2-Fab-hA20 scFv-AD2- Okt3 (22)-3s CD22 C_(H)1-DDD2-Fab-hLL2scFv-AD2- Okt3 (C2)-3s HLA-DR C_(H)1-DDD2-Fab-hL243 scFv-AD2- Okt3(M1)-3s MUC5AC C_(H)1-DDD2-Fab-hPAM4 scFv-AD2- Okt3 (14)-3s CEACAM5C_(H)1-DDD2-Fab-hMN-14 scFv-AD2- Okt3 (15)-3s CEACEAM6C_(H)1-DDD2-Fab-hMN-15 scFv-AD2- Okt3 (E1)-3s TROP-2C_(H)1-DDD2-Fab-hRS7 scFv-AD2- Okt3

Analytical Methods—

Size-exclusion high-performance liquid chromatography (SE-HPLC) wasperformed with an Alliance HPLC System with a BIOSUITE™ 250, 4-m UHR SECcolumn (Waters Corp). Electrospray ionization time of flight (ESI-TOF)liquid chromatography/mass spectrometry (LC-MS) was performed with a1200-series HPLC coupled with a 6210 TOF MS (Agilent Technologies, SantaClara, Calif.). The (19)-3s was resolved by reversed phase HPLC(RP-HPLC)at 60° C., using a 14-min gradient of 30-80% acetonitrile in 0.1%aqueous formic acid with an Aeris widepore 3.6 μm C4 column(Phenomenex). For the TOF MS, the capillary and fragmentor voltages wereset to 5500 and 300 V, respectively.

Cell Lines and Reagents—

Raji, Ramos, Daudi, LS174T and Capan-1 cell lines were purchased fromthe American Type Cell Culture Collection (ATCC, Manassas, Md.) andNalm-6 cells were purchased from Deutsche Sammlung von Mikroorganismenand Zellinien (DSMZ, Braunchweig, Germany). All cell lines, exceptCapan-1, were maintained in RPMI-1640 containing 10% FBS, 1%L-glutamine, 1% penicillin-streptomycin and 1% MEM nonessential aminoacids. Capan-1 cells were maintained with 20% FBS. All cell culturemedia and supplements were purchased from Life Technologies (Carlsbad,Calif.).

PBMCs and T Cell Isolation—

Human peripheral blood mononuclear cells (PBMC) were purified from wholedonor blood (Blood Center of NJ, East Orange, N.J.) using UNI-SEP_(MAXI)tubes (Novamed, Ltd, Jerusalem, Israel). CD3-positive T cells wereisolated from PBMCs by negative selection using the Pan T Cell IsolationKit (Miltenyi Biotec, Auburn, Calif.), according to the manufacturer'sprotocol. Efficiency of T cell isolation was assessed by FACS afterstaining the enriched T cells with anti-CD3-PE antibody. In some cases,further staining with CD-19 and CD-14 was performed as well to identifycontaminating cells.

T Cell Activation—

Isolated T cells were plated in 6-well tissue culture plates at a finaldensity of 2.25×10⁶ cells/well. Daudi cells were added to some wells ata final density of 1.5×10⁶ cells/well, other wells were left to containonly T cells. Alternatively, PBMCs were added to 6-well tissue cultureplates at a final cell density of 6×10⁶ cells/well. The volume of eachwell was brought up to 3 mL. To the appropriate wells, 3 ng/mL of(19)-3s, (M1)-3s or (19)-DDD2 was added. After incubation overnight at37° C., 1 mL of each sample was removed. The cells were pelleted andlabeled on ice with CD69-APC and CD3-PE for 20 minutes. Cells werewashed 2 times with 1% BSA in PBS and analyzed using a FACSCALIBER™ flowcytometer (BD Biosciences, San Jose, Calif.).

T Cell Proliferation—

PBMCs were seeded in T25 flasks at a concentration of 1×10⁶ cells/mLcontaining the specified reagents. For B cell-depleted flasks, B cellswere removed by negative selection using a B cell isolation kit fromMiltenyi according to manufacturer's protocol. On select days, 100 μL ofmedia was removed from each flask, labeled with anti-CD7-APC for 20minutes on ice, washed once and resuspended in 300 μL of 1% BSA/PBScontaining 7-AAD. For each sample, the entire volume is analyzed using aFACSCALIBER™ flow cytometer. Each sample is counted in duplicate.Analysis is performed using FlowJo Software. For each sample, dead(7-AAD+) cells, and debris (based on forward vs. side scatter) wasremoved. Finally, live CD7+ cells were selected and plotted using Prismsoftware.

Cell Binding Assays (Jurkat/Capan-1)—

Jurkat cells were stained with PKH26 Red Fluorescent Cell Linker Kit(Sigma) according to manufacturer's protocol. Capan-1 cells were stainedwith 5 μM CFSE (carboxyfluorescein diacetate succinimidyl ester, LifeTechnologies) according to manufacturer's protocol. Labeled Capan-1cells were added to 8-well chamber slides (ThermoWaltham, Mass.) andallowed to attach overnight. The following day, media was removed andPKH26-labeled Jurkat cells were added in media containing 0.1 μg/mL of(E1)-3s, (M1)-3s or (19)-3s. Following a 1-hour incubation at 37° C.,slides were washed with PBS to remove any unbound cells and observed byfluorescence microscopy.

Cell Binding Assays (Jurkat/Daudi)—

Jurkat and Daudi cells were labeled with anti-CD3-PE and anti-CD20-FITC,respectively. Labeled cells were then coincubated at a 2.5:1 ratio with0.1 μg/mL (19)-3s for 30 minutes at room temperature. Aliquots of cellswere then observed by fluorescence microscopy.

Cytotoxicity Assay (Hematologic Tumor Cell Lines)—

Target cells were labeled with PKH67 Green Fluorescent Cell Linker Kit(Sigma) according to the manufacturer's protocol. Briefly, 5×10⁶ targetcells were resuspended in 250 μL of diluent C. In a second tube 1 μL ofPKH26 dye is added to 250 μL of diluent C. The cell suspension is thenadded to the dye solution, mixed thoroughly and incubated at RT for 2minutes. The reaction was quenched by adding an equal volume of FBS. Thelabeled cells were then washed 3 times with complete RPMI. Unstimulated,isolated T cells were used as effector cells. Effector cells andPKH67-labeled target cells were combined at a 10:1 ratio and plated in48-well plates containing serial dilutions of (19)-3s or (14)-3s. Eachwell contained 5×10⁴ target cells and 5×10⁵ effector cells. Jeko-1assays were performed in 20% RPMI. Plates were incubated for 18-24 hoursin a 37° C. incubator containing 5% CO₂. Following incubation, all cellswere removed from 48-well plates into flow cytometer tubes andresuspended in 1% BSA/PBS containing 1 ug/mL of 7AAD, to distinguishlive from dead cells, and 30,000 COUNTBRIGHT™ Absolute Counting Beads(Life Technologies). Cells were analyzed on a FACSCALIBER™ flowcytometer. For each sample, 8,000 COUNTBRIGHT™ beads were counted as anormalized reference. Data were analyzed using FlowJo software(Treestar, Inc., Ashland, Oreg.). For each sample, dead cells and debriswere excluded and total live target cells were counted.

Cytotoxicity Assay (Solid Tumor Cell Lines)—

Target cells were labeled with PKH67 Green Fluorescent Cell Linker Kit(Sigma) following the same procedure as for staining with PKH23.Effector cells used were as follows: For Capan-1 assays, CD8+ enriched Tcells were used, following purification from a CD8+ enrichment column(R&D Systems, Minneapolis, Minn.). For LS174T cells: Stimulated T cellswere used after incubation of PBMC for 5 days in media containing 25U/mL IL-2 and 50 ng/mL Okt3 Mab, followed by 2 days incubation in mediacontaining 25 U/mL IL-2 alone. Effector cells and PKH67-labeled targetcells were combined at a 3:1 ratio (5×10⁴ target cells and 1.5×10⁵effector cells/well) and plated over 48-well plates containing serialdilutions of (E1)-3s, (14)-3s or (19)-3s. Capan-1 assays were performedin 20% RPMI. Plates were incubated for 42-48 hours in a 37° C. incubatorcontaining 5% CO₂. Following incubation, suspension cells were combinedwith trypsinized attached cells from all wells and transferred into flowcytometer tubes. Cells were washed one time and resuspended in 1%BSA/PBS containing 1 ug/mL of 7AAD, to distinguish live from dead cells,and 30,000 COUNTBRIGHT™ Absolute Counting Beads. Cells were analyzed ona FACSCALIBER™ flow cytometer. For each sample, 8,000 COUNTBRIGHT™ beadswere counted as a normalized reference. Data were analyzed using FlowJosoftware (Treestar, Inc., Ashland, Oreg.). For each sample, dead cellsand debris were excluded and total live target cells were counted.

In Vivo Efficacy—

Female NOD/SCID mice, 8 weeks old, were purchased from Charles River(Wilmington, Mass.). Mice were injected s.c. with a mixture of Raji(1×10⁶) and human PBMCs (5×10⁶ cells) mixed 1:1 with matrigel. Therapybegan 1 hour later. Treatment regimens, dosages, and number of animalsin each experiment are described in the Results. Animals were monitoreddaily for signs of tumor out-growth. Once tumors appeared, they weremeasured twice weekly. Tumor volume (TV) was determined by measurementsin two dimensions using calipers, with volumes defined as: L×w²/2, whereL is the longest dimension of the tumor and w the shortest. Efficacy wasdetermined by a log-rank test using Prism GraphPad software (v5;LaJolla, Calif.) on Kaplan-Meier curves using survival surrogateendpoints as time for tumor progression (TTP) to 1.0 cm³. Significancewas considered at P<0.05.

Results

Construction and Biochemical Analysis of T-Cell Redirecting BispecificAntibodies.

The DNL™ method was used to generate a panel of (X)-3s, T-cellredirecting bsAbs for targeting of various tumor-associated antigensincluding CD19, CD20, HLA-DR, TROP-2, CEACAM5 and MUC5AC. The purity ofthese structures was demonstrated by SE-HPLC and SDS-PAGE analysis,where only bands representing the three constituent polypeptides(Okt3scFv-AD2, hA19-Fd-DDD2 and hA19 kappa) were evident (data notshown). LC-MS analysis identified a single RP-HPLC peak having adeconvoluted mass spectrum consistent (mass accuracy=11 ppm) with thecalculated mass (137432.37 Da) of (19)-3s from its deduced amino acidsequence, including the predicted amino-terminal pyroglutamates on theOkt3scFv-AD2 and each of the two C_(H)1-DDD2-hA19 Fd chains (data notshown). No additional post-translational modifications, includingglycosylation were indicated.

Immune Synapse Formation Between Daudi Burkitt Lymphoma and T Cells,Mediated by (19)-3s.

The effects of the T-cell redirecting (19)-3s DNL™ complex on targetingeffector T cells to CD19⁺ lymphoma cells was examined (FIG. 2). Freshlyisolated T cells were combined with Daudi cells at an E:T ratio of2.5:1. Cells were treated with 0, 1 or 5 μg/mL of (19)-3s DNL™ complexfor 30 min at room temperature prior to analysis by flow cytometry.Anti-CD20-FITC and anti-CD7-APC were used to identify Daudi and T cells,respectively. Co-binding was indicated as the % of CD20⁺/CD7⁺ events.After treatment with (19)-3s, 45.5% of flow events were CD20/CD7dual-positive, indicating synapsed Daudi and T cells (FIG. 2A), comparedto 2% measured for the mixed cells without antibody (FIG. 2B). Additionof (19)-3s resulted in association of >90% of the Daudi with T cells(FIG. 2C). These results show that the (19)-3s DNL™ complex waseffective to direct T cells to the targeted antigen-expressing lymphomacells.

Synapse formation between T cells and target lymphoma cells wasdemonstrated by fluorescence microscopy (FIG. 3) Jurkat (T cells) andDaudi (B cells) were combined at a 1:1 ratio, treated with 0.1 μg/mL ofthe (19)-3s DNL™ complex for 30 minutes and stained with anti-CD20-FITC(FIG. 3A) and anti-CD3-PE (FIG. 3B), prior to analysis by fluorescencemicroscopy. The merged image (FIG. 3C) reveals synapse formation betweengreen-stained Daudi and red-stained Jurkat cells. Synapse formation wasnot evident in the absence of (19)-3s (FIG. 3D). FIG. 3C demonstratesthat the target lymphoma cells are in direct contact with the targeted Tcells.

A dose-response series was performed for (19)-3s mediated association ofT cells to an exemplary B-cell lymphoma line (FIG. 4). As shown in FIG.4, under the conditions of this experiment, saturation of(19)-3s-mediated cell-to-cell association of T cells to target cells wasreached at a concentration between 0.037 and 0.111 μg/ml of the DNL™complex.

FIG. 5 shows a comparison of the relative efficacies of BITE™ (FIG. 5A),DART™ (FIG. 5A) and DNL™ (FIG. 5B) anti-CD3×anti-CD19 complexes forredirecting T cells to targeted CD19⁺ B cells. The data for BITE™ andDART™ was obtained from Moore et al. (2011, Blood 117:4542-51). At thelowest concentration tested of 0.0005 μg/ml, the (19)-3s DNL™ complexwas more effective than BITE™ or DART™ at targeting T cells to B-celllymphoma (FIG. 5). The (19)-3s DNL™ complex also induced a slightlyhigher maximum level of cell-to-cell association than the comparableBITE™ and DART™ complexes (FIG. 5A). Although difficult to extrapolatefrom the single data points generated for the (19)-3s DNL™ complex, theEC₅₀ levels appeared to be similar for BITE™, DART™ and DNL™ (FIG. 5).

(19)-3s, (E1)-3s and (M1)-3s-Mediated Cell-Cell Association of T Cellsto Target Tumor Cells.

To evaluate the ability of the T-cell redirecting BsAbs to facilitatethe association of T cells to their target tumor cells, Jurkat T cellswere coincubated with target tumor cells containing (X)-3s and evaluatedby flow cytometry and fluorescence microscopy. Jurkat T cells are a CD4+T cell leukemia line, chosen for their ability to demonstrate T cellbinding without depletion of the FITC labeled Daudi cells in thepresence of various concentrations of (19)-3s and analyzed by flowcytometry for the detection of double positive (CD3+CD20+) populationsindicating T cell-B cell associated complexes. An apparent cell-cellassociation was seen following treatment with 0.5 ng/mL of (19)-3s andafter treatment with 0.1 μg/mL over 25% of the cell population existedin a cell-cell association (FIG. 5). Fluorescent microscopy supportsthis data, as immune synapses are evident following treatment with 0.1μg/mL (19)-3s (FIG. 4). No synapse formation was seen in the absence of(19)-3s (data not shown).

This cell-cell association was observed in the pancreatic tumor lineCapan-1 as well (FIG. 6). Capan-1 expresses high levels of TROP2 andmoderate levels of MUC5AC. Therefore, both the TROP2-targeting bsAb,(E1)-3s (FIG. 6C), and MUC5AC-targeting bsAb, (M1)-3s (FIG. 6B) werecompared to the non-targeting control bsAb, (19)-3s (FIG. 6A).CFSE-labeled Capan-1 cells were coincubated with PKH26-labeled Jurkat inthe presence of these bsAbs. Fluorescent microscopy revealed, asexpected, large T-cell/Capan complexes mediated by (E1)-3s, followed bysmaller, yet substantial complexes mediated by (M1)-3s and relativelylow complex formation following (19)-3s treatment (FIG. 6).

(19)-3s Specifically Induces T Cell Activation and Proliferation.

The ability of (19)-3s to activate T cells was evaluated either in PBMCs(FIG. 7A), or T cells coincubated with Daudi B cells (FIG. 7B), bymeasuring the expression levels of CD69, an early marker of T cellactivation. Treatment with 3 ng/mL of (19)-3s induced T cell activationin T cells coincubated with Daudi B cells as indicated by a >50-foldincrease in CD69 expression compared with non-targeting controlantibodies, (19)-DDD2 and (M1)-3s, as well as T cells treated with(19)-3s without Daudi target cells (FIG. 7B). Similar results wereobserved when the antibodies were incubated with PBMCs, containing bothT and B cells; (19)-3s stimulated CD69 expression levels >20-fold higherthan non-targeting controls (FIG. 7A). In the absence of target cells,purified T cells treated with (19)-3s did not show activation (FIG. 7C).

T cell proliferation, as another indication of T cell activation, wasevaluated after treatment of PBMCs with various CD3-targetingantibodies. (19)-3s at 3 nM or 30 pM induced T cell proliferationsimilar to that of the positive control IL-2/PHA (FIG. 8A).Non-targeting control antibody, (14)-3s, shows some non-specific T cellproliferation at the highest (3 nM) concentration (FIG. 8A). However, Tcell proliferation was not observed in PBMCs depleted of B cells (FIG.8B), suggesting that target cells are necessary for specific (19)-3sinduced T cell proliferation.

(X)-3s Re-Directed T-Cell Mediated Killing of Malignant Cell Lines.

The cytotoxicity of each T-cell targeting molecule was evaluated by itsability to mediate lysis of specific tumor target cells. For thehematologic tumor cell lines, a 10:1 E:T ratio using an unstimulated,enriched T cell population as the effector cells in an 18-24 hour assaydemonstrated the optimal assay conditions. The CD19-targeting bsAb,(19)-3s induced the most potent specific killing of the relatively lowCD19-expressing cell lines Ramos (IC₅₀=0.17 pM, Lysis_(Max)=79%) Daudi(IC₅₀=1 pM, Lysis_(Max)=60%), and Nalm6 (IC₅₀=6 pM, Lysis_(Max)=93%)(FIG. 9A). Interestingly, the high CD19-expressing cell lines, Namalwa(IC₅₀=63 pM, Lysis_(Max)=60%) and Raji (IC₅₀=3 nM, Lysis_(Max)=41%) werethe least sensitive to (19)-3s (FIG. 9A). The non-targeting (14)-3s DNL™construct had little cytotoxic effect in any of the cell lines tested(FIG. 9B). Consistent cytotoxic effects of the (19)-3s construct on theNalm-6 ALL cell line were obtained with PBMCs obtained from twodifferent donors (FIG. 9C).

The in vitro cytotoxic effects of (20)-3s, (22)-3s and (C2)-3s T-cellredirecting bsAbs were determined in several cell lines (FIG. 10). TheCD22-targeting bsAb, (22)-3s, demonstrated potent (IC₅₀=5 pM,Lysis_(Max)=60%) specific T-cell mediated lysis in the CD22-positiveDaudi cell line (FIG. 10C), but not in the CD22-negative Namalwa cells(FIG. 10A).

The CD20-targeting bsAb, (20)-3s demonstrated the highest potency in thehigher-expressing CD20 cell lines, Daudi (IC₅₀=<0.3 pM, Lysis_(Max)=90%)(FIG. 10C) and Jeko (IC₅₀=1 pM, Lysis_(Max)=90%) (FIG. 10B), compared tothe lower CD20-expressing Namalwa cell line (IC₅₀=30 pM,Lysis_(Max)=53%) (FIG. 10A).

The HLA-DR-targeting bsAb, (C2)-3s was tested in the HLA-DR expressingJeko-1 cell line (IC₅₀=20 pM, Lysis_(Max)=88%) (FIG. 10B).

At an E:T ratio of 10:1, using isolated T cells as effector cells, thebsAbs induced potent T cell-mediated cytotoxicity in various B cellmalignancies, including Burkitt lymphoma (Daudi, Ramos, Namalwa) mantlecell lymphoma (Jeko-1) and acute lymphoblastic leukemia (Nalm-6) (Table7). A non-tumor binding control, (14)-3s, induced only moderate T-cellkilling at >10 nM. The nature of the antigen/epitope, particularly itssize and proximity to the cell surface, appears to be more importantthan antigen density for T-cell retargeting potency (Table 7). It islikely that (20)-3s is consistently more potent than (19)-3s and(C2)-3s, even when the expression of CD19 or HLA-DR is considerablyhigher than CD20, as seen with Namalwa and Jeko-1, respectively (Table7). This is likely because the CD20 epitope comprises a smallextracellular loop having close proximity to the cell surface. Whencompared directly using Daudi, (22)-3s was the least potent. Compared toCD19 and CD20, CD22 is expressed at the lowest density, is a rapidlyinternalizing antigen, and its epitope is further away from the cellsurface. Each of these factors may contribute to its reduced potency.Finally, sensitivity to T-cell retargeted killing iscell-line-dependent, as observed using (19)-3s, where Raji (IC₅₀>3 nM)is largely unresponsive yet Ramos (IC₅₀=2 pM) is highly sensitive, eventhough the former expresses higher CD19 antigen density (Table 7).

In conclusion, (19)-3s, (20)-3s, (22)-3s and (C2)-3s bind to T cells andtarget B cells simultaneously and induce T-cell-mediated killing invitro. The modular nature of the DNL method allowed the rapid productionof several related conjugates for redirected T-cell killing of various Bcell malignancies, without the need for additional recombinantengineering and protein production. The close proximity of the CD20extracellular epitope to the cell surface resulted in the highestpotency for (20)-3s.

TABLE 7 Ex vivo re-directed T-cell killing Antigen Expression² IC₅₀ ⁴(pM) Cell Line Type¹ CD19 CD20 CD22 HLA- (19)-3s (20)- (22)-3s (C2)-Daudi BL 1.00 1.00 1.00 1.00 1 0.3 6 N.D. Ramos BL 0.76 0.65 0.26 0.36 20.4 N.D.  2 Nalm-6 ALL 1.63 0.05 0.19 0.17 6 N.D. N.D. N.D. Namalwa BL0.76 0.11 0.05 0.40 63 30 >3000 N.D. Raji BL 1.41 0.69 0.59 0.84 >3000N.D. N.D. N.D. Jeko-1 MCL 0.89 1.02 0.05 1.06 3000 1 N.D. 20 ¹BL,Burkitt lymphoma; ALL, acute lymphoblastic leukemia; MCL, mantle celllymphoma. ²Expression level determined by flow cytometry and normalizedto that of Daudi. ³IC₅₀, the picomolar concentration that achieved 50%target cell killing.

The in vitro cytotoxic effects of T-cell redirecting bsAbs were alsodetermined in solid tumor cells (FIG. 11). For the solid tumor celllines, optimal assay conditions were determined to be a 3:1 E:T ratiousing stimulated T cells in a 42-48 hour assay. Each bsAb inducedspecific T-cell mediated lysis of the tumor target cells. TheCEACAM5-expressing colon adenocarcinoma cell line, LS-174T, demonstratedpotent specific lysis (IC₅₀=2 pM) following treatment with (14)-3s (FIG.11A). (E1)-3s mediated potent specific lysis of the TROP2 expressingCapan-1 pancreatic adenocarcinoma cell line (IC₅₀=29 pM) (FIG. 11B). Thegastric carcinoma cell line NCI-N87, which expresses high levels of bothCEACAM6 and TROP2 demonstrated very potent specific lysis to both T-celltargeting molecules, (15)-3s and (E1)-3s (IC₅₀=3 pM and 0.85 pMrespectively) (FIG. 11C). The non-targeting control antibody, (19)-3s,induced low (<20%) non-specific lysis at concentrations >1 nM forCapan-1 and LS174T, and moderate (˜40%) non-specific lysis in NCI-N87cells (FIG. 11A-C). A summary of the in vitro cytotoxicity data forvarious T-cell redirecting bsAbs in a variety of tumor cell lines isshown in FIG. 12. The various constructs showed a maximal cell lysis ofup to 90% or more of the targeted tumor cells, with IC₅₀ values for celllines expressing the targeted antigen that were generally in the lowpicomolar range (FIG. 12).

Example 2 In Vivo Studies of T-Cell Redirecting DNL™ Complex

One potential limitation of small (<60 kDa) scFv-based constructs, suchas BITE™ and DART™, is the requirement for administration by long-termcontinuous infusion, due to their toxicity and rapid clearance fromcirculation. Because the molecular size of DNL™ bsAbs is above thethreshold typically associated with renal clearance, it should exhibitslower clearance from circulation. We measured the pharmacokineticparameters in mice following a single bolus i.v. injection of 5 mg/kg ofthe (19)-3s bsAb (data not shown). A biphasic clearance was observedwith a t½α and t½β of 1.1 and 5.1 h, respectively, resulting in an areaunder the curve of 1880 pmol*h/mL (data not shown), which was nearly6-fold greater than that reported for MT103 (anti-CD19×anti-CD3 BITE™)administered at the same molar concentration (US PatentUS2010/0303827A1). The major difference is apparently a longer Wm for(19)-3s (data not shown). Because of the potentially advantageousproperties of (19)-3s, we evaluated the possibility of using lessfrequent dosing schedules rather than daily dosing, which is typicallyused for BITE™ in animal studies.

A pilot study was performed using Raji human Burkitt lymphoma xenograftsin NOD/SCID mice reconstituted with human PBMCs (FIG. 13, FIG. 14). Rajicells (1×10⁶ cells/mouse) were combined with freshly isolated PBMCs(5×10⁶ cells/mouse) from a single healthy donor, mixed 1:1 withmatrigel, and injected SC into all of the animals in the study on Day 0.Groups of 5 mice received i.v. injections of (19)-3s totaling 130 μg asa single dose on Day 0 (FIG. 13B), three doses of 43 μg (Days 0, 2 and4) (FIG. 13C) or five daily doses of 26 μg (Days 0-5) (FIG. 13D). Theuntreated group (FIG. 13A), which was inoculated with the same cellmixture but did not receive (19)-3s, had a median survival time (MST) of31 days. Each therapy regimen improved survival (P≦0.05), with the threedose (every other day) schedule providing the greatest survival benefit(MST=91 days; P=0.0018 by log-rank analysis).

A follow up study was begun to determine the efficacy of less frequentdosing (FIG. 14). Groups of 9 NOD/SCID mice were inoculated with Rajiand PBMCs in a similar fashion as above. In this study, therapy wasextended to two weeks, compared to one week in the first study. Groupsreceived i.v. injections of (19)-3s totaling 360 μg as 2×130-μg (FIG.14B), 4×65-μg (FIG. 14D) or 6×43-μg doses over two weeks (FIG. 14E). Anadditional group was administered 2×130-μg doses SC, instead of i.v.(FIG. 14C). For comparison, control groups of untreated mice (FIG. 14A)or mice treated with non-targeting (M1)-3s antibody (FIG. 14F) wereprepared. As of Day 28, each of the (19)-3s treatment groups hadsignificantly smaller AUC than the untreated control (P<0.05).Surprisingly, two weekly doses via the SC route was apparently aseffective as greater frequency i.v. dosing.

In vivo studies were also performed using solid tumors (FIG. 15).NOD/SCID mouse xenografts were prepared as described above, for theLS174T colon adenocarcinoma (FIG. 15A, FIG. 15B) or Capan-1 pancreaticcarcinoma (FIG. 15C, FIG. 15D). In each case, mice administered thetargeting (E1)-3s (FIG. 15B) or (14)-3s (FIG. 15D) bsAb DNL™ constructsshowed improved survival compared to controls.

In conclusion, the T-cell retargeting bsAbs, including (19)-3s, (E1)-3sand (M1)-3s DNL™ constructs, mediated synapse formation between T cellsand B cells, colon adenocarcinoma or pancreatic carcinoma cells,respectively, via monovalent and bivalent binding to CD3 and CD19,respectively. T-cell activation, proliferation and target cell killingwere induced by the DNL™ bsAbs at pM concentrations in an ex vivosetting. Advantageous properties of the DNL™ bsAbs, including bivalenttumor binding and slower clearance, would allow for less frequent dosingand possibly SC administration, compared to BITE™ or DART™ constructs,which are administered i.v. daily in animal models and as a continuousinfusion in the clinic. The modular nature of the DNL™ method allows therapid production of a large number of related conjugates for redirectedT-cell killing of various malignancies, without the need for additionalrecombinant engineering and protein production.

The person of ordinary skill in the art will realize that otherantibodies that bind to CD3, CD19 or other disease-associated antigensare known in the art and any such antibody can be used to make F(ab)₂,scFv or other antibody fragments using techniques well known in the art.Such alternative antibodies or fragments thereof may be utilized in theinstant methods and compositions. As discussed below, methods of makingDOCK-AND-LOCK™ (DNL™) complexes may be applied to incorporate any knownantibodies or antibody fragments into a stable, physiologically activecomplex.

Example 3 Interferon-α Enhances the Cytotoxic Effect of T-CellRedirecting Bispecific Antibodies

The therapeutic efficacy of the (E1)-3s anti-TROP-2×anti-CD3 bispecificantibody, made from hRS7 and OKT3 as a DNL™ complex, was tested for itsability to delay tumor outgrowth of Capan-1 human pancreaticadenocarcinoma tumor cells when mixed with human T-cells and injectedinto mice. The effect of interferon-α (either in the form of E1*-2b orPEGASYS) when combined with this therapy was also evaluated.

Methods

Five week-old female NOD/SCID mice were injected s.c. with a mixture ofCapan-1 (5×10⁶) and human T-cells (2.5×10⁶ cells) mixed 1:1 withmatrigel (E:T ratio of 1:2). There were six different treatment groupsof 8 mice each. Treatment consisted of one group receiving 47 μg (E1)-3si.v. every day for five days starting 1 hour after the administration ofthe Capan-1/T-cell mixture. Two groups were treated with equimolaramounts of IFN, one received the DNL molecule made fromIFN-α2b-DDD2-CK-hRS7 IgG1 (E1*-2b; 2.5 μg s.c. weekly×4 wks) whileanother received PEGASYS (Roche; 0.6 μg s.c. weekly×4 wks). Two othergroups received a combination of (E1)-3s plus E1*2b or (E1)-3s plusPEGASYS. The final group control group remained untreated. Table 7summarizes the various treatment groups.

TABLE 7 Treatment Groups for (E1)-3s Therapy (E1)-3s Therapy of a HumanPancreatic Carcinoma Xenograft (Capan-1) in NOD/SCID Mice Group (N)Amount Injected Schedule 1 8 Untreated N.A. 2 8 (E1)-3s qdx5 (47 μgi.v.) 3 8 E1*-2b qwkx4 (2.5 μg s.c.) 4 8 PEGASYS qwkx4 (0.6 μg s.c.) 5 8(E1)-3s + qdx5 + E1*-2b qwkx4 6 8 (E1)-3s + qdx5 + PEGASYS qwkx4

Mice were monitored daily for signs of tumor out-growth. All animals hadtheir tumors measured twice weekly once tumors began to come up. Micewere euthanized for disease progression if their tumor volumes exceeded1.0 cm³ in size.

Results

Mean tumor volumes for the various groups are shown in FIG. 16. The datacontaining PEGASYS® groups (FIG. 16B) are shown on a separate graph fromthe E1*2b groups (FIG. 16A) for clarity. All treatments weresignificantly better at controlling tumor growth in terms ofarea-under-the-curve (AUC) when compared to the untreated mice out today 29, which was when the first mouse in the untreated group waseuthanized for disease progression (P<0.0009; AUC_(29 days)). Combining(E1)-3s with PEGASYS® resulted in the best anti-tumor response overallin terms of tumor out-growth (FIG. 16B). This treatment wassignificantly better than any of the individual treatments (P<0.042;AUC) as well as superior to the combination of (E1)-3s plus E1*-2b(P=0.0312; AUC_(53 days)) (FIG. 16A). The combination of (E1)-3s plusE1*2b could significantly control tumor growth when compared to E1*2b orPEGASYS® alone (P<0.0073; AUC_(46 days)) but not (E1)-3s alone (FIG.16A-B). There were no significant differences between mice treated with(E1)-3s, PEGASYS®, or E1*-2b (FIG. 16A-B).

In terms of survival, all treatments provide a significant survivalbenefit when compared to the untreated mice (P<0.0112; log-rank) (FIG.17). As of day 81, there was no significant difference in mediansurvival times (MST) between mice treated with the combination of(E1)-3s plus E1*-2b and those treated (E1)-3s plus PEGASYS® (MST=79.5and >81 days, respectively) (FIG. 17). The mice treated with (E1)-3splus PEGASYS® had a significantly improved survival outcome than any ofthe individual treatments (P<0.0237) (FIG. 17). Mice treated with(E1)-3s plus E1*2b had a survival benefit when compare to mice treatedwith either E1*-2b or PEGASYS® alone (MST=53 days for both; P<0.0311)but not when compared to mice treated with just (E1)-3s (MST=68 days)(FIG. 17). Treatment with (E1)-3s provided a significant improvement insurvival when compared to mice treated with E1*-2b (P=0.0406) but notwhen compared to mice treated with PEGASYS® alone (FIG. 17). There wasno significant differences between mice treated with only E1*2b andthose treated with PEGASYS® alone (FIG. 17).

The results demonstrate that addition of interferon-α provides asubstantial increase in survival and decrease in tumor growth whencombined with a T-cell redirecting bsAb. The person of ordinary skillwill realize that the improved efficacy observed with addition of type Ior type III interferons (interferon-α, interferon-β, or interferon-λ) isnot limited to the specific (E1)-3s bsAb, but will be observed withother T-cell redirecting bsAbs, made either as DNL™ complexes or inother forms, such as BITE™ or DART™.

Example 4 General Techniques for DOCK-AND-LOCK™

The general techniques discussed below may be used to generate DNL™complexes with AD or DDD moieties attached to any antibodies orantigen-binding antibody fragments, using the disclosed methods andcompositions.

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (V_(H) and V_(L)) sequences.Using molecular biology tools known to those skilled in the art, theseIgG expression vectors can be converted into Fab-DDD or Fab-ADexpression vectors.

To generate Fab-DDD expression vectors, the coding sequences for thehinge, CH2 and CH3 domains of the heavy chain were replaced with asequence encoding the first 4 residues of the hinge, a 14 residue linkerand a DDD moiety, such as the first 44 residues of human RIIα (referredto as DDD1, SEQ ID NO:1). To generate Fab-AD expression vectors, thesequences for the hinge, CH2 and CH3 domains of IgG were replaced with asequence encoding the first 4 residues of the hinge, a 15 residue linkerand an AD moiety, such as a 17 residue synthetic AD called AKAP-IS(referred to as AD1, SEQ ID NO:3), which was generated usingbioinformatics and peptide array technology and shown to bind RIIαdimers with a very high affinity (0.4 nM). See Alto, et al. Proc. Natl.Acad. Sci., U.S.A (2003), 100:4445-50. Two shuttle vectors were designedto facilitate the conversion of IgG-pdHL2 vectors to either Fab-DDD1 orFab-AD1 expression vectors, as described below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC, SEQ ID NO:102) followed byfour glycines and a serine, with the final two codons (GS) comprising aBamHI restriction site. The 410 bp PCR amplimer was cloned into thePGEMT® PCR cloning vector (PROMEGA®, Inc.) and clones were screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of DDD1 preceded by 11 residues of the linker peptide, with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 103) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, whichoverlap by 30 base pairs on their 3′ ends, were synthesized and combinedto comprise the central 154 base pairs of the 174 bp DDD1 sequence. Theoligonucleotides were annealed and subjected to a primer extensionreaction with Taq polymerase. Following primer extension, the duplex wasamplified by PCR. The amplimer was cloned into PGEMT® and screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 104) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective PGEMT® shuttle vector.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 (SEQ ID NO:2) appended to the carboxyl terminus of theFd of hMN-14 via a 14 amino acid residue Gly/Ser peptide linker. Thefusion protein secreted is composed of two identical copies of hMN-14Fab held together by non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide and residues 1-13 of DDD2, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair to C-DDD2-Fab-hMN-14.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2 (SEQID NO:4) appended to the carboxyl terminal end of the CH1 domain via a14 amino acid residue Gly/Ser peptide linker. AD2 has one cysteineresidue preceding and another one following the anchor domain sequenceof AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Generation of TF2 DNL™ Construct

A trimeric DNL™ construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Production of TF10 DNL™ Construct

A similar protocol was used to generate a trimeric TF10 DNL™ construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The TF10 bispecific ([hPAM4]₂×h679) antibody was producedusing the method disclosed for production of the (anti CEA)₂×anti HSGbsAb TF2, as described above. The TF10 construct bears two humanizedPAM4 Fabs and one humanized 679 Fab.

The two fusion proteins (hPAM4-DDD2 and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD2. The reaction mixture was incubated at room temperaturefor 24 hours under mild reducing conditions using 1 mM reducedglutathione. Following reduction, the reaction was completed by mildoxidation using 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP291-affigel resin, which binds with highspecificity to the h679 Fab.

Example 5 Production of AD- and DDD-Linked Fab and IgG Fusion Proteinsfrom Multiple Antibodies

Using the techniques described in the preceding Example, the IgG and Fabfusion proteins shown in Table 8 were constructed and incorporated intoDNL™ constructs. The fusion proteins retained the antigen-bindingcharacteristics of the parent antibodies and the DNL™ constructsexhibited the antigen-binding activities of the incorporated antibodiesor antibody fragments.

TABLE 8 Fusion proteins comprising IgG or Fab Fusion Protein BindingSpecificity C-AD1-Fab-h679 HSG C-AD2-Fab-h679 HSG C-(AD)₂-Fab-h679 HSGC-AD2-Fab-h734 Indium-DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DRC-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1 IGF-1RC-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5

Example 6 Production and Use of a DNL™ Construct Comprising TwoDifferent Antibody Moieties and a Cytokine

In certain embodiments, trimeric DNL™ constructs may comprise threedifferent effector moieties, for example two different antibody moietiesand a cytokine moiety. We report here the generation andcharacterization of a bispecific MAb-IFNα, designated 20-C2-2b, whichcomprises two copies of IFN-α2b and a stabilized F(ab)₂ of hL243(humanized anti-HLA-DR; IMMU-114) site-specifically linked to veltuzumab(humanized anti-CD20). In vitro, 20-C2-2b inhibited each of fourlymphoma and eight myeloma cell lines, and was more effective thanmonospecific CD20-targeted MAb-IFNα or a mixture comprising the parentalantibodies and IFNα in all but one (HLA-DR⁻/CD20⁻) myeloma line (notshown), suggesting that 20-C2-2b is useful for the treatment of varioushematopoietic disorders. The 20-C2-2b displayed greater cytotoxicityagainst KMS12-BM (CD20⁺/HLA-DR⁺ myeloma) than monospecific MAb-IFNα thattargets only HLA-DR or CD20 (not shown), indicating that all threecomponents in 20-C2-2b can contribute to toxicity.

Antibodies

The abbreviations used in the following discussion are: 20(C_(H)3-AD2-IgG-v-mab, anti-CD20 IgG DNL™ module); C2(C_(H)1-DDD2-Fab-hL243, anti-HLA-DR Fab₂ DNL™ module); 2b (dimericIFNβ2B-DDD2 DNL™ module); 734 (anti-in-DTPA IgG DNL™ module used asnon-targeting control). The following MAbs were provided byImmunomedics, Inc.: veltuzumab or v-mab (anti-CD20 IgG₁), hL243γ4p(Immu-114, anti-HLA-DR IgG₄), a murine anti-IFNα MAb, and ratanti-idiotype MAbs to v-mab (WR2) and hL243 (WT).

DNL™ Constructs

Monospecific MAb-IFNα (20-2b-2b, 734-2b-2b and C2-2b-2b) and thebispecific HexAb (20-C2-C2) were generated by combination of anIgG-AD2-module with DDD2-modules using the DNL™ method, as described inthe preceding Examples. The 734-2b-2b, which comprises tetrameric IFNα2band MAb h734 [anti-Indium-DTPA IgG1], was used as a non-targetingcontrol MAb-IFNα.

The construction of the mammalian expression vector as well as thesubsequent generation of the production clones and the purification ofC_(H)3-AD2-IgG-v-mab are disclosed in the preceding Examples. Theexpressed recombinant fusion protein has the AD2 peptide linked to thecarboxyl terminus of the C_(H)3 domain of v-mab via a 15 amino acid longflexible linker peptide. Co-expression of the heavy chain-AD2 and lightchain polypeptides results in the formation of an IgG structure equippedwith two AD2 peptides. The expression vector was transfected into Sp/ESFcells (an engineered cell line of Sp2/0) by electroporation. The pdHL2vector contains the gene for dihydrofolate reductase, thus allowingclonal selection, as well as gene amplification with methotrexate (MTX).Stable clones were isolated from 96-well plates selected with mediacontaining 0.2 μM MTX. Clones were screened for C_(H)3-AD2-IgG-vmabproductivity via a sandwich ELISA. The module was produced in rollerbottle culture with serum-free media.

The DDD-module, IFNα2b-DDD2, was generated as discussed above byrecombinant fusion of the DDD2 peptide to the carboxyl terminus of humanIFNα2b via an 18 amino acid long flexible linker peptide. As is the casefor all DDD-modules, the expressed fusion protein spontaneously forms astable homodimer.

The C_(H)1-DDD2-Fab-hL243 expression vector was generated fromhL243-IgG-pdHL2 vector by excising the sequence for theC_(H)1-Hinge-C_(H)2-C_(H)3 domains with SacII and EagI restrictionenzymes and replacing it with a 507 bp sequence encoding C_(H)1-DDD2,which was excised from the C-DDD2-hMN-14-pdHL2 expression vector withthe same enzymes. Following transfection of C_(H)1-DDD2-Fab-hL243-pdHL2into Sp/ESF cells by electroporation, stable, MTX-resistant clones werescreened for productivity via a sandwich ELISA using 96-well microtiterplates coated with mouse anti-human kappa chain to capture the fusionprotein, which was detected with horseradish peroxidase-conjugated goatanti-human Fab. The module was produced in roller bottle culture.

Roller bottle cultures in serum-free H-SFM media and fed-batchbioreactor production resulted in yields comparable to other IgG-AD2modules and cytokine-DDD2 modules generated to date.C_(H)3-AD2-IgG-v-mab and IFNα2b-DDD2 were purified from the culturebroths by affinity chromatography using MABSELECT™ (GE Healthcare) andHIS-SELECT® HF Nickel Affinity Gel (Sigma), respectively, as describedpreviously (Rossi et al., Blood 2009, 114:3864-71). The culture brothcontaining the C_(H)1-DDD2-Fab-hL243 module was applied directly toKAPPASELECT® affinity gel (GE-Healthcare), which was washed to baselinewith PBS and eluted with 0.1 M Glycine, pH 2.5.

Generation of 20-C2-2b by DNL™

Three DNL™ modules (C_(H)3-AD2-IgG-v-mab, C_(H)1-DDD2-Fab-hL243, andIFN-α2b-DDD2) were combined in equimolar quantities to generate thebsMAb-IFNα, 20-C2-2b. Following an overnight docking step under mildreducing conditions (1 mM reduced glutathione) at room temperature,oxidized glutathione was added (2 mM) to facilitate disulfide bondformation (locking). The 20-C2-2b was purified to near homogeneity usingthree sequential affinity chromatography steps. Initially, the DNL™mixture was purified with Protein A (MABSELECT™), which binds theC_(H)3-AD2-IgG-v-MAb group and eliminates un-reacted IFNα2b-DDD2 orC_(H)1-DDD2-Fab-hL243. The Protein A-bound material was further purifiedby IMAC using HIS-SELECT® HF Nickel Affinity Gel, which bindsspecifically to the IFNα2b-DDD2 moiety and eliminates any constructslacking this group. The final process step, using an hL243-anti-idiotypeaffinity gel removed any molecules lacking C_(H)1-DDD2-Fab-hL243.

The skilled artisan will realize that affinity chromatography may beused to purify DNL™ complexes comprising any combination of effectormoieties, so long as ligands for each of the three effector moieties canbe obtained and attached to the column material. The selected DNL™construct is the one that binds to each of three columns containing theligand for each of the three effector moieties and can be eluted afterwashing to remove unbound complexes.

The following Example is representative of several similar preparationsof 20-C2-2b. Equimolar amounts of C_(H)3-AD2-IgG-v-mab (15 mg),C_(H)1-DDD2-Fab-hL243 (12 mg), and IFN-α2b-DDD2 (5 mg) were combined in30-mL reaction volume and 1 mM reduced glutathione was added to thesolution. Following 16 h at room temperature, 2 mM oxidized glutathionewas added to the mixture, which was held at room temperature for anadditional 6 h. The reaction mixture was applied to a 5-mL Protein Aaffinity column, which was washed to baseline with PBS and eluted with0.1 M Glycine, pH 2.5. The eluate, which contained ˜20 mg protein, wasneutralized with 3 M Tris-HCl, pH 8.6 and dialyzed into HIS-SELECT®binding buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH₂PO₄, pH 8.0)prior to application to a 5-mL HIS-SELECT® IMAC column. The column waswashed to baseline with binding buffer and eluted with 250 mM imidazole,150 mM NaCl, 50 mM NaH₂PO₄, pH 8.0.

The IMAC eluate, which contained ˜11.5 mg of protein, was applieddirectly to a WP (anti-hL243) affinity column, which was washed tobaseline with PBS and eluted with 0.1 M glycine, pH 2.5. The processresulted in 7 mg of highly purified 20-C2-2b. This was approximately 44%of the theoretical yield of 20-C2-2b, which is 50% of the total startingmaterial (16 mg in this example) with 25% each of 20-2b-2b and 20-C2-C2produced as side products.

Generation and Characterization of 20-C2-2b

The bispecific MAb-IFNα was generated by combining the IgG-AD2 module,C_(H)3-AD2-IgG-v-mab, with two different dimeric DDD-modules,C_(H)1-DDD2-Fab-hL243 and IFNβ2b-DDD2. Due to the random association ofeither DDD-module with the two AD2 groups, two side-products, 20-C2-C2and 20-2b-2b are expected to form, in addition to 20-C2-2b.

Non-reducing SDS-PAGE (not shown) resolved 20-C2-2b (˜305 kDa) as acluster of bands positioned between those of 20-C2-C2 (˜365 kDa) and20-2b-2b (255 kDa). Reducing SDS-PAGE resolved the five polypeptides(v-mab HC-AD2, hL243 Fd-DDD2, IFNα2b-DDD2 and co-migrating v-mab andhL243 kappa light chains) comprising 20-C2-2b (not shown). IFNα2b-DDD2and hL243 Fd-DDD2 are absent in 20-C2-C2 and 20-2b-2b. MABSELECT™ bindsto all three of the major species produced in the DNL™ reaction, butremoves any excess IFNα2b-DDD2 and C_(H)1-DDD2-Fab-hL243. TheHIS-SELECT® unbound fraction contained mostly 20-C2-C2 (not shown). Theunbound fraction from WT affinity chromatography comprised 20-2b-2b (notshown). Each of the samples was subjected to SE-HPLC andimmunoreactivity analyses, which corroborated the results andconclusions of the SDS-PAGE analysis.

Following reduction of 20-C2-2b, its five component polypeptides wereresolved by RP-HPLC and individual ESI-TOF deconvoluted mass spectrawere generated for each peak (not shown). Native, but notbacterially-expressed recombinant IFNα2, is O-glycosylated at Thr-106(Adolf et al., Biochem J 1991; 276 (Pt 2):511-8). We determined that˜15% of the polypeptides comprising the IFNα2b-DDD2 module areO-glycosylated and can be resolved from the non-glycosylatedpolypeptides by RP-HPLC and SDS-PAGE (not shown). LC/MS analysis of20-C2-2b identified both the O-glycosylated and non-glycosylated speciesof IFNα2b-DDD2 with mass accuracies of 15 ppm and 2 ppm, respectively(not shown). The observed mass of the O-glycosylated form indicates anO-linked glycan having the structure NeuGc-NeuGc-Gal-GalNAc, which wasalso predicted (<1 ppm) for 20-2b-2b (not shown). LC/MS identified bothv-mab and hL243 kappa chains as well as hL243-Fd-DDD2 (not shown) assingle, unmodified species, with observed masses matching the calculatedones (<35 ppm). Two major glycoforms of v-mab HC-AD2 were identified ashaving masses of 53,714.73 (70%) and 53,877.33 (30%), indicating G0F andG1F N-glycans, respectively, which are typically associated with IgG(not shown). The analysis also confirmed that the amino terminus of theHC-AD2 is modified to pyroglutamate, as predicted for polypeptideshaving an amino terminal glutamine.

SE-HPLC analysis of 20-C2-2b resolved a predominant protein peak with aretention time (6.7 min) consistent with its calculated mass and betweenthose of the larger 20-C2-C2 (6.6 min) and smaller 20-2b-2b (6.85 min),as well as some higher molecular weight peaks that likely representnon-covalent dimers formed via self-association of IFNα2b (not shown).

Immunoreactivity assays demonstrated the homogeneity of 20-C2-2b witheach molecule containing the three functional groups (not shown).Incubation of 20-C2-2b with an excess of antibodies to any of the threeconstituent modules resulted in quantitative formation of high molecularweight immune complexes and the disappearance of the 20-C2-2b peak (notshown). The HIS-SELECT® and WT affinity unbound fractions were notimmunoreactive with WT and anti-IFNα, respectively (not shown). TheMAb-IFNα showed similar binding avidity to their parental MAbs (notshown).

IFNα Biological Activity

The specific activities for various MAb-IFNα were measured using acell-based reporter gene assay and compared to peginterferon alfa-2b(not shown). Expectedly, the specific activity of 20-C2-2b (2454IU/pmol), which has two IFNα2b groups, was significantly lower thanthose of 20-2b-2b (4447 IU/pmol) or 734-2b-2b (3764 IU/pmol), yetgreater than peginterferon alfa-2b (P<0.001) (not shown). The differencebetween 20-2b-2b and 734-2b-2b was not significant. The specificactivity among all agents varies minimally when normalized to IU/pmol oftotal IFNα. Based on these data, the specific activity of each IFNα2bgroup of the MAb-IFNα is approximately 30% of recombinant IFNα2b (4000IU/pmol).

In the ex-vivo setting, the 20-C2-2b DNL™ construct depleted lymphomacells more effectively than normal B cells and had no effect on T cells(not shown). However, it did efficiently eliminate monocytes (notshown). Where v-mab had no effect on monocytes, depletion was observedfollowing treatment with hL243α4p and MAb-IFNα, with 20-2b-2b and734-2b-2b exhibiting similar toxicity (not shown). Therefore, thepredictably higher potency of 20-C2-2b is attributed to the combinedactions of anti-HLA-DR and IFNα, which may be augmented by HLA-DRtargeting. These data suggest that monocyte depletion may be apharmacodynamic effect associated anti-HLA-DR as well as IFNα therapy;however, this side effect would likely be transient because the monocytepopulation should be repopulated from hematopoietic stem cells.

The skilled artisan will realize that the approach described here toproduce and use bispecific immunocytokine, or other DNL™ constructscomprising three different effector moieties, may be utilized with anycombinations of antibodies, antibody fragments, cytokines or othereffectors that may be incorporated into a DNL™ construct, for examplethe combination of anti-CD3 and anti-CD19 or other anti-TAA with IFNα2b.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and used without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the METHODS described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A method of inducing a T-cell mediated cytotoxic immune response against a Trop-2 positive human cancer cell comprising: a) administering a T-cell redirecting complex, comprising at least one binding site for human CD3 and at least one binding site for human Trop-2, to a subject to induce a T-cell mediated immune response against the Trop-2 positive human cancer cell and wherein the T-cell redirecting complex comprises: (i) a first antibody moiety conjugated to an AD (anchoring domain) moiety from an AKAP protein; and (ii) a second antibody moiety conjugated to a DDD (dimerization and docking domain) moiety, wherein the amino acid sequence of said DDD moiety is residues 1-44 of human protein kinase A (PKA) RIIα wherein two copies of the DDD moiety form a dimer that binds to one copy of the AD moiety to form the complex after “human cancer cell”.
 2. The method of claim 1, further comprising administering interferon-α to the subject.
 3. The method of claim 2, wherein the combination of interferon and T-cell redirecting complex is more effective than interferon alone and T-cell redirecting complex alone.
 4. The method of claim 2, wherein the interferon is administered before, simultaneously with, or after the T-cell redirecting complex.
 5. The method of claim 2, wherein the interferon is administered as free interferon, PEGylated interferon, an interferon fusion protein or interferon conjugated to an antibody.
 6. The method of claim 1, wherein the T-cell redirecting complex is a bispecific antibody comprising a first antibody moiety that binds to CD3 and a second antibody moiety that binds to Trop-2.
 7. The method of claim 6, wherein the first and second antibody moieties are selected from the group consisting of an scFv, a Fab and a dAb.
 8. The method of claim 6, wherein the second antibody moiety is hRS7.
 9. The method of claim 1, wherein the T-cell redirecting complex is administered intravenously.
 10. The method of claim 1, wherein the T-cell redirecting complex is administered subcutaneously. 